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. 2019 Jul 3;8:e42695. doi: 10.7554/eLife.42695

Table 1. Sequences of constructs used in the study.

Construct Sequence Notes
Ezrin ISHMHHHHHHHHHHKCKVYEPVSYHVQESLQDEGAEPTGYSAELSSEGIRDDRNEEKRITEAEKNERVQRQLLTLSSELSQARDENKRTHNDIIHNENMRQGRDKYKTLRQIRQGNTKQRIDEFEAL Human, Ezrin actin binding domain, residues 477–586, with N-terminal His10 - KCK fusion.
Grb2 GPLGSMEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNV Human, residues 1–217.
Grb2Basic GPLGSMEAIAKYDFKATADDELSFKRGDILKVLNEECDQNWYKAELNGKDGFIPKNYIEMKPHPWFFGKIPRAKAEEMLSKQRHDGAFLIRESESAPGDFSLSVKFGNDVQHFKVLRDGAGKYFLWVVKFNSLNELVDYHRSTSVSRNQQIFLRDIEQVPQQPTYVQALFDFDPQEDGELGFRRGDFIHVMDNSDPNWWKGACHGQTGMFPRNYVTPVNRNVKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKA Human, residues 1–217 with the basic region of N-WASP (x2) fused to the C-terminal.
LAT GGSLEHHHHHHHHGIQFKRPHTVAPWPPAFPPVTSFPPLSQPDLLPIPRSPQPLGGSHRTPSSRRDSDGANSVASFENEEPACEDADEDEDDFHNPGYLVVLPDSTPATSTAAPSAPALSTPGIRDSAFSMESIDDYVNVPESGESAEASLDGSREYVNVSQELHPGAAKTEPAALSSQEAEEVEEEGAPDYENLQELN Human, residues 48–233 (short isoform) with His8N-terminal fusion. This construct only contains the four C-terminal Tyr residues (Y132, Y171, Y191, and Y226) that are sufficient for TCR signaling. Y171, Y191, and Y226 are the three tyrosines that are recognized by Grb2 when phosphorylated. All other tyrosine residues were mutated to Phe residues.
Nck GHMCMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 1–377, with mutations C139S, C232A, C266S, C340S. A single Cys was added to the N-terminus.
His-Nck (FL) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 1–377, with N-terminal His8-GGSC(GGS)4 fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck (FLΔL1) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSGGSAGGSAGGSATLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 2–377, with N-terminal His8-GGSC(GGS)4fusion and C139S, C232A, C266S, and C340S mutations. ARKASIVKNLKD in the N-terminal portion of L1 has been replaced with GGSAGGSAGGSA.
His-Nck
(L1-S2-L2-S3-L3-SH2)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 59–377 (No S1), with N-terminal His8-GGSC(GGS)4 fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck (L1(KtoE)-S2-L2-S3-L3-SH2) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSAREASIVENLEDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 59–377 (No S1), with N-terminal His8-GGSC(GGS)4fusion and C139S, C232A, C266S, and C340S mutations. The basic N-terminal portion of L1 has the following basic to acidic mutations: K64E, K69E, and K72E.
His-Nck (L1(HM)-S2-L2-S3-L3-SH2) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEKNSARKASNSKNSKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 59–377, with N-terminal His8-GGSC(GGS)4fusion and C139S, C232A, C266S, and C340S mutations. Additional Hydrophobic Mutations in L1: I67N, V68S, L71S.
His-Nck
(S1-L1-S3-L3-SH2)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 2–108 and 191–377, with N-terminal His8-GGSC(GGS)4 fusion and C232A, C266S, and C340S mutations.
His-Nck
(S1-L1-S2-L2-S3)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQN Human, residues 2–252, with N-terminal His8-GGSC(GGS)4 fusion and C139S and C232A mutations.
His-Nck
(S2-L2-S3-L3-SH2)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGXEGXFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 106–377, with N-terminal His8-GGSC(GGS)4fusion and C139S, C232A, C266S, and C340S mutations.
His-Nck
(S3-L3-SH2)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 190–377, with N-terminal His8-GGSC(GGS)4fusion and C232A, C266S, and C340S mutations.
His-Nck
(L3-SH2)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLENPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 253–377, with N-terminal His8-GGSC(GGS)4fusion and C266S and C340S mutations.
His-Nck (S1) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSGSAAAS Human, residues 2–61, with N-terminal His8-GGSC(GGS)4 fusion.
His-Nck (S2) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDGSAAAS Human, residues 106–165, with N-terminal His8-GGSC(GGS)4fusion and C139S mutation.
His-Nck (S3) GHMHHHHHHHHGGSCGGSGGSGGSGGSLEQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNGSAAAS Human, residues 190–252, with N-terminal His8-GGSC(GGS)4fusion and C232A mutation.
His-NckAcidic
(FL(L1 KtoE))		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSAREASIVENLEDTLGIGKVKRKPSVPDSASPADDSFVDPGERLYDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 2–377, with C139S, C232A, C266S, and C340S mutations. The basic N-terminal portion of L1 has the following basic to acidic mutations: K64E, K69E, and K72E.
His-NckBasic
(FL(L1 basic))		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERKNSARKASIVKNLKDTLGIGKVKRKPSVPKSASPADKSFVKPGKRLYKLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKSSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKARKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQSDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYSIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human, residues 2–377, with C139S, C232A, C266S, and C340S mutations. The acidic C-terminal portion of L1 has the following acidic to basic mutations: D88K, D95K, D99K, E102K, and D106K.
His-NckNeutral
(FL(L1 GGSA)10)		 GHMHHHHHHHHGGSCGGSGGSGGSGGSLEMAEEVVVVAKFDYVAQQEQELDIKKNERLWLLDDSKSWWRVRNSMNKTGFVPSNYVERGGSAGGSAGGSAGGSAGGSAGSGGSAGGSAGGSAGGSAGGSAGSDLNMPAYVKFNYMAEREDELSLIKGTKVIVMEKCSDGWWRGSYNGQVGWFPSNYVTEEGDSPLGDHVGSLSEKLAAVVNNLNTGQVLHVVQALYPFSSSNDEELNFEKGDVMDVIEKPENDPEWWKCRKINGMVGLVPKNYVTVMQNNPLTSGLEPSPPQCDYIRPSLTGKFAGNPWYYGKVTRHQAEMALNERGHEGDFLIRDSESSPNDFSVSLKAQGKNKHFKVQLKETVYCIGQRKFSTMEELVEHYKKAPIFTSEQGEKLYLVKHLS Human full-length Nck with L1 replaced by a GGSA linker, residues 1–58, 109–377, with an N-terminal His8-GGSC(GGS)4 fusion and C139S, C232A, C266S, and C340S mutations.
N-WASP GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505.
His-N-WASP MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His6 fusion.
N-WASPBasic GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505. Basic Region (186-200) has been doubled.
His-N-WASPBasic MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTKEKKKGKAKKKRLTKGKEKKKGKAKKKRITKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His6 fusion. Basic Region (186-200) has been doubled.
N-WASPNeutral GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYA
ADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTGGSGGSGGSGGSGGSADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED 		Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505. Basic Region (186-200) has been removed and replaced with (GGS)5.
His-N-WASPNeutral MGHHHHHHDYDIPTTENLYFQGSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGKKCVTMSSAVVQLYAADRNCMWSKKCSGVACLVKDNPQRSYFLRIFDIKDGKLLWEQELYNNFVYNSPRGYFHTFAGDTCQVALNFANEEEAKKFRKAVTDLLGRRQRKSEKRRDPPNGPNLPMATVDIKNPEITTNRFYGPQVNNISHTGGSGGSGGSGGSGGSADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISEAQLKDRETSKVIYDFIEKTGGVEAVKNELRRQAPPPPPPSRGGPPPPPPPPHNSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPSVAVPPPPPNRMYPPPPPALPSSAPSGPPPPPPSVLGVGPVAPPPPPPPPPPPGPPPPPGLPSDGDHQVPTTAGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVADGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDEDDEEDFEDDDEWED Human WIP N-WASP EVH1 binding motif, residues 451–485 fused to human N-WASP, residues 26–505 with N-terminal His6 fusion. Basic Region (186-200) has been removed and replaced with (GGS)5.
N-WASP BPVCA GSEFKEKKKGKAKKKRAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPHPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIREGAQLKKVEQNSRPVSCSGRDALLDQIRQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWED Rat, N-WASP fusion of basic, proline-rich, and VCA domains, residues 183–193 and 273–501.
N-WASP BPVCA (R/A R/A) GSEFKEKKKGKAKKKRAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPHPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVPASSGNKAALLDQIAEGAQLKKVEQNSRPVSCSGRDALLDQIAQGIQLKSVSDGQESTPPTPAPTSGIVGALMEVMQKRSKAIHSSDEDEDDDDEEDFEDDDEWED Rat, N-WASP fusion of basic, proline-rich, and VCA domains, residues 183–193 and 273–501 with R410A and R438A mutations.
N-WASP BP GSEFKEKKKGKAKKKRLTKADIGTPSNFQHIGHVGWDPNTGFDLNNLDPELKNLFDMCGISAPPPPPPSRGGPPPPPPPPHSSGPPPPPARGRGAPPPPPSRAPTAAPPPPPPSRPGVVVPPPPPNRMYPPPPPALPSSAPSGPPPPPPLSMAGSTAPPPPPPPPPPPGPPPPPGLPSDGDHQVP Rat, N-WASP fusion of basic, proline-rich, residues 183–239 and 273–396.
SLP-76 GHMDNGGWSSFEEDDYESPNDDQDGEDDGDYESPNEEEEAPVEDDADYEPPPSNDEEALQNSILPAKPFPNSNSMFIDRPPSGKTPQQPPVPPQRPMAALPPPPAGRNHSPLPPPQTNHEEPSRSRNHKTAKLPAPSIDRSTKPPLDRSLAPFDREPFTLGKKPPFSDKPSIPAGRSLGEHLPKIQKPPLPPTTERHERSSPLPGKKPPVPKHGWGPDRRENDEDDVHQRPLPQPALLPMSSNTFPSRSTKPSPMNPLSSHMPGAFSESNSSFPQSASLPPFFSQGPSNRPPIRAEGRNFPLPLPNKPRPPSPAEEENCSLNEGSLEVLFQ Human, residues 101–420, with a Cys added near the C-terminus for labeling.
Sos1 GPLGSNDTVFIQVTLPHGPRSASVSSISLTKGTDEVPVPPPVPPRRRPESAPAESSPSKIMSKHLDSPPAIPPRQPTSKAYSPRYSISDRTSISDPPESPLLPPREPVRTPDVFSSSPLHLQPPPLGKKSDHGNAFFPNSPSPFTPPPPQTPSPHGTRRHLPSPPLTQEVDLHSIAGPPVPPRQSTSQHIPKLPPKTYKREHTHPSMC Human, poly-proline rich region, residues 1117–1319 with a Cys added at the C-terminus for labeling.
WASP GSESRFYFHPISDLPPPEPYVQTTKSYPSKLARNESRENESLFTFLGRKCLTLATAVVQLYLALPPGAEHWTKEHCGAVCFVKDNPQKSYFIRLYGLQAGRLLWEQELYSQLVYSTPTPFFHTFAGDDCQAGLNFADEDEAQAFRALVQEKIQKRNQRQSGDRRQLPPPPTPANEERRGGLPPLPLHPGGDQGGPPVGPLSLGLATVDIQNPDITSSRYRGLPAPGPSPADKKRSGKKKISKADIGAPSGFKHVSHVGWDPQNGFDVNNLDPDLRSLFSRAGISEAQLTDAETSKLIYDFIEDQGGLEAVRQEMRRQEPLPPPPPPSRGGNQLPRPPIVGGNKGRSGPLPPVPLGIAPPPPTPRGPPPPGRGGPPPPPPPATGRSGPLPPPPPGAGGPPMPPPPPPPPPPPSSGNGPAPPPLPPALVPAGGLAPGGGRGALLDQIRQGIQLNKTPGAPESSALQPPPQSSEGLVGALMHVMQKRSRAIHSSDEGEDQAGDEDEDDEWDD Human WIP WASP EVH1 binding motif, residues 451–485 fused to human WASP, residues 39–502.