Interaction Details


Details of experiments between P31947 and Q9Y4H2
Note that the results of all experiments are listed, regardless of the modification states of the fragments.


Experiment series 1

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1159-1168
Site: 14-3-3_pSer1164
Modification: (SEP)1164
Sequence: TPVSPSFAHN
Construct: TPVSP(pSer)FAHN (crude synthetic peptide)
Average holdup BI: 0
Holdup BI standard deviation: 0.03
Immobilized partner concentration (10-6M): 113
P value: 0.03
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 2

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1159-1168
Site: 14-3-3_pSer1164
Modification: (SEP)1164
Sequence: TPVSPSFAHN
Construct: TPVSP(pSer)FAHN (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.06
Immobilized partner concentration (10-6M): 142
P value: 0.3
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 3

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1159-1168
Site: 14-3-3_pSer1164
Modification: (SEP)1164
Sequence: TPVSPSFAHN
Construct: TPVSP(pSer)FAHN (crude synthetic peptide)
Average holdup BI: -0.07
Holdup BI standard deviation: 0.05
Immobilized partner concentration (10-6M): 111
P value: 1.65
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 4

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1171-1180
Site: 14-3-3_pSer1176
Modification: (SEP)1176
Sequence: RHNSASVENV
Construct: RHNSA(pSer)VENV (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.11
Immobilized partner concentration (10-6M): 113
P value: 1.13
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 5

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1171-1180
Site: 14-3-3_pSer1176
Modification: (SEP)1176
Sequence: RHNSASVENV
Construct: RHNSA(pSer)VENV (crude synthetic peptide)
Average holdup BI: -0.02
Holdup BI standard deviation: 0.34
Immobilized partner concentration (10-6M): 111
P value: 0.05
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 6

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1181-1190
Site: 14-3-3_pSer1186
Modification: (SEP)1186
Sequence: SLRKSSEGGV
Construct: SLRKS(pSer)EGGV (crude synthetic peptide)
Average holdup BI: -0.09
Holdup BI standard deviation: 0.19
Immobilized partner concentration (10-6M): 113
P value: 0.4
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 7

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1278-1287
Site: 14-3-3_pSer1283
Modification: (SEP)1283
Sequence: PGDKSSWGRT
Construct: PGDKS(pSer)WGRT (crude synthetic peptide)
Average holdup BI: 0.05
Holdup BI standard deviation: 0.08
Immobilized partner concentration (10-6M): 113
P value: 0.56
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 8

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1278-1287
Site: 14-3-3_pSer1283
Modification: (SEP)1283
Sequence: PGDKSSWGRT
Construct: PGDKS(pSer)WGRT (crude synthetic peptide)
Average holdup BI: -0.03
Holdup BI standard deviation: 0.08
Immobilized partner concentration (10-6M): 142
P value: 0.37
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 9

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 1278-1287
Site: 14-3-3_pSer1283
Modification: (SEP)1283
Sequence: PGDKSSWGRT
Construct: PGDKS(pSer)WGRT (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.12
Immobilized partner concentration (10-6M): 111
P value: 0.06
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 10

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 341-350
Site: 14-3-3_pSer346
Modification: (SEP)346
Sequence: RSRTDSLAAT
Construct: RSRTD(pSer)LAAT (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.07
Immobilized partner concentration (10-6M): 113
P value: 1.78
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 11

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 341-350
Site: 14-3-3_pSer346
Modification: (SEP)346
Sequence: RSRTDSLAAT
Construct: RSRTD(pSer)LAAT (crude synthetic peptide)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.03
Immobilized partner concentration (10-6M): 142
P value: 1.19
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 12

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 341-350
Site: 14-3-3_pSer346
Modification: (SEP)346
Sequence: RSRTDSLAAT
Construct: RSRTD(pSer)LAAT (crude synthetic peptide)
Average holdup BI: -0.22
Holdup BI standard deviation: 0.21
Immobilized partner concentration (10-6M): 111
P value: 1.36
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 13

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 379-388
Site: 14-3-3_pSer384
Modification: (SEP)384
Sequence: GARPVSVAGS
Construct: GARPV(pSer)VAGS (crude synthetic peptide)
Average holdup BI: 0.09
Holdup BI standard deviation: 0.04
Immobilized partner concentration (10-6M): 113
P value: 2.38
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 14

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 379-388
Site: 14-3-3_pSer384
Modification: (SEP)384
Sequence: GARPVSVAGS
Construct: GARPV(pSer)VAGS (crude synthetic peptide)
Average holdup BI: 0.17
Holdup BI standard deviation: 0.04
Immobilized partner concentration (10-6M): 142
P value: 4.37
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 15

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 379-388
Site: 14-3-3_pSer384
Modification: (SEP)384
Sequence: GARPVSVAGS
Construct: GARPV(pSer)VAGS (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.06
Immobilized partner concentration (10-6M): 111
P value: 1.53
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 16

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 427-436
Site: 14-3-3_pSer432
Modification: (SEP)432
Sequence: HSRSMSMPVA
Construct: HSRSM(pSer)MPVA (crude synthetic peptide)
Average holdup BI: 0.81
Holdup BI standard deviation: 0.06
Immobilized partner concentration (10-6M): 113
P value: 2.44
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
Measured pKd: 4.58
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 17

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 427-436
Site: 14-3-3_pSer432
Modification: (SEP)432
Sequence: HSRSMSMPVA
Construct: HSRSM(pSer)MPVA (crude synthetic peptide)
Average holdup BI: 0.79
Holdup BI standard deviation: 0.03
Immobilized partner concentration (10-6M): 142
P value: 5.35
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
Measured pKd: 4.43
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 18

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 427-436
Site: 14-3-3_pSer432
Modification: (SEP)432
Sequence: HSRSMSMPVA
Construct: HSRSM(pSer)MPVA (crude synthetic peptide)
Measured dissociation constant (10-6M): <100
Immobilized partner concentration (10-6M): 111
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 1
Measured pKd: 4
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 19

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 518-527
Site: 14-3-3_pSer523
Modification: (SEP)523
Sequence: SNTPESIAET
Construct: SNTPE(pSer)IAET (crude synthetic peptide)
Average holdup BI: 0.38
Holdup BI standard deviation: 0.23
Immobilized partner concentration (10-6M): 113
P value: 1.73
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
Measured pKd: 3.74
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 20

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 518-527
Site: 14-3-3_pSer523
Modification: (SEP)523
Sequence: SNTPESIAET
Construct: SNTPE(pSer)IAET (crude synthetic peptide)
Average holdup BI: -0.06
Holdup BI standard deviation: 0.21
Immobilized partner concentration (10-6M): 142
P value: 0.29
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 21

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 518-527
Site: 14-3-3_pSer523
Modification: (SEP)523
Sequence: SNTPESIAET
Construct: SNTPE(pSer)IAET (crude synthetic peptide)
Average holdup BI: 0.15
Holdup BI standard deviation: 0.13
Immobilized partner concentration (10-6M): 111
P value: 1.19
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 22

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 555-564
Site: 14-3-3_pSer560
Modification: (SEP)560
Sequence: SYRRVSGDAA
Construct: SYRRV(pSer)GDAA (crude synthetic peptide)
Average holdup BI: -0.32
Holdup BI standard deviation: 0.53
Immobilized partner concentration (10-6M): 113
P value: 0.73
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 23

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 555-564
Site: 14-3-3_pSer560
Modification: (SEP)560
Sequence: SYRRVSGDAA
Construct: SYRRV(pSer)GDAA (crude synthetic peptide)
Average holdup BI: -0.08
Holdup BI standard deviation: 0.93
Immobilized partner concentration (10-6M): 142
P value: 0.07
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 24

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 555-564
Site: 14-3-3_pSer560
Modification: (SEP)560
Sequence: SYRRVSGDAA
Construct: SYRRV(pSer)GDAA (crude synthetic peptide)
Average holdup BI: -0.2
Holdup BI standard deviation: 0.42
Immobilized partner concentration (10-6M): 111
P value: 0.48
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 25

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 603-612
Site: 14-3-3_pSer608
Modification: (SEP)608
Sequence: ATFSGSAGRL
Construct: ATFSG(pSer)AGRL (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.02
Immobilized partner concentration (10-6M): 113
P value: 4.21
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 26

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 603-612
Site: 14-3-3_pSer608
Modification: (SEP)608
Sequence: ATFSGSAGRL
Construct: ATFSG(pSer)AGRL (crude synthetic peptide)
Average holdup BI: 0.12
Holdup BI standard deviation: 0.04
Immobilized partner concentration (10-6M): 142
P value: 3.31
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 27

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 603-612
Site: 14-3-3_pSer608
Modification: (SEP)608
Sequence: ATFSGSAGRL
Construct: ATFSG(pSer)AGRL (crude synthetic peptide)
Average holdup BI: 0.08
Holdup BI standard deviation: 0.02
Immobilized partner concentration (10-6M): 111
P value: 3.15
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 28

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 718-727
Site: 14-3-3_pSer723
Modification: (SEP)723
Sequence: RTFPASGGGY
Construct: RTFPA(pSer)GGGY (crude synthetic peptide)
Average holdup BI: 0.11
Holdup BI standard deviation: 0.03
Immobilized partner concentration (10-6M): 113
P value: 3.37
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 29

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 718-727
Site: 14-3-3_pSer723
Modification: (SEP)723
Sequence: RTFPASGGGY
Construct: RTFPA(pSer)GGGY (crude synthetic peptide)
Average holdup BI: 0.13
Holdup BI standard deviation: 0.04
Immobilized partner concentration (10-6M): 142
P value: 3.83
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 30

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 718-727
Site: 14-3-3_pSer723
Modification: (SEP)723
Sequence: RTFPASGGGY
Construct: RTFPA(pSer)GGGY (crude synthetic peptide)
Average holdup BI: 0.1
Holdup BI standard deviation: 0.02
Immobilized partner concentration (10-6M): 111
P value: 3.68
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 31

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 725-734
Site: 14-3-3_pSer730
Modification: (SEP)730
Sequence: GGYKASSPAE
Construct: GGYKA(pSer)SPAE (crude synthetic peptide)
Average holdup BI: -0.03
Holdup BI standard deviation: 0.04
Immobilized partner concentration (10-6M): 113
P value: 0.78
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 32

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 725-734
Site: 14-3-3_pSer730
Modification: (SEP)730
Sequence: GGYKASSPAE
Construct: GGYKA(pSer)SPAE (crude synthetic peptide)
Average holdup BI: -0.03
Holdup BI standard deviation: 0.12
Immobilized partner concentration (10-6M): 142
P value: 0.27
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 33

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 725-734
Site: 14-3-3_pSer730
Modification: (SEP)730
Sequence: GGYKASSPAE
Construct: GGYKA(pSer)SPAE (crude synthetic peptide)
Average holdup BI: 0.01
Holdup BI standard deviation: 0.07
Immobilized partner concentration (10-6M): 111
P value: 0.12
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 34

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 730-739
Site: 14-3-3_pSer735
Modification: (SEP)735
Sequence: SSPAESSPED
Construct: SSPAE(pSer)SPED (crude synthetic peptide)
Average holdup BI: 0.03
Holdup BI standard deviation: 0.6
Immobilized partner concentration (10-6M): 113
P value: 0.03
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 35

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 730-739
Site: 14-3-3_pSer735
Modification: (SEP)735
Sequence: SSPAESSPED
Construct: SSPAE(pSer)SPED (crude synthetic peptide)
Average holdup BI: 0.06
Holdup BI standard deviation: 0.79
Immobilized partner concentration (10-6M): 111
P value: 0.05
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
The affinity was below the detection threshold of the assay.
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 36

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 574-583
Site: 14-3-3_pThr579
Modification: (TPO)579
Sequence: RTYSLTTPAR
Construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Average holdup BI: 0.99
Holdup BI standard deviation: 0.01
Immobilized partner concentration (10-6M): 142
P value: 8.43
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2021-10-13
Number of measurements: 5
Measured pKd: 5.76
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 5))

Experiment series 37

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 574-583
Site: 14-3-3_pThr579
Modification: (TPO)579
Sequence: RTYSLTTPAR
Construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Average holdup BI: 0.98
Holdup BI standard deviation: 0
Immobilized partner concentration (10-6M): 113
P value: 8.25
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
Measured pKd: 5.65
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))

Experiment series 38

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 574-583
Site: 14-3-3_pThr579
Modification: (TPO)579
Sequence: RTYSLTTPAR
Construct: biotin-ttds-RTYSL(pThr)TPAR (HPLC-purified synthetic peptide)
Measured dissociation constant (10-6M): 8.6
Standard deviation of dissociation constant (10-6M): 1
Experiment method: competitive Fluorescence Polarization
Details of affinity fitting: Competitive binding equation in ProFit
Date of measurement: 2022-02-09
Number of measurements: 3
Measured pKd: 5.06
DOI: 10.1101/2022.12.08.519103

Experiment series 39

Protein A

Protein: SFN
Fragment: 1-248
Site: 14-3-3
Modification: 100_uM_FCA
Construct: avi-his6-MBP-TEVsite-14-3-3sigma

Protein B

Protein: IRS2
Fragment: 574-583
Site: 14-3-3_pThr579
Modification: (TPO)579
Sequence: RTYSLTTPAR
Construct: RTYSL(pThr)TPAR (crude synthetic peptide)
Average holdup BI: 0.98
Holdup BI standard deviation: 0
Immobilized partner concentration (10-6M): 111
P value: 8.85
Experiment method: HU Multiplex
Details of affinity fitting: hyperbolic binding equation
Date of measurement: 2022-02-17
Number of measurements: 4
Measured pKd: 5.64
DOI: 10.1101/2022.12.08.519103
Experimental Details

-log10(P Two-sided unpaired T-test (4 vs 4))