| Reagent type . | Designation . | Source or reference . | Additional information . |
|---|---|---|---|
| Cell line | HEK293T | University of California, San Francisco, Cell Culture Facility | Lentivirus package |
| Cell line | Jurkat T cell E6.1 | University of California, San Francisco, Cell Culture Facility | Stable expression cell line construction |
| Cell line | PLCG1 null | This study (J74B) | PLCG1 knockout by CRISPR |
| Cell line | GRB2 null | This study (J81) | GRB2 knockout by CRISPR |
| Cell line | GADS null | This study (J82) | GADS knockout by CRISPR |
| Plasmid | pMD2.G | Vale Laboratory (XSB395) | Viral packaging, plasmid |
| Plasmid | poPAX | Vale Laboratory (XSB396) | Viral packaging, plasmid |
| Plasmid | pSpCas9(BB)-PLCG1 234F-2A-GFP | This study (XSB717) | CRISPR-cas9 vector for knocking out PLCG1 |
| Plasmid | pSpCas9(BB)-Grb2 42F-2A-GFP | This study (XSB740) | CRISPR-cas9 vector for knocking out GRB2 |
| Plasmid | pSpCas9(BB)-Gads 97F-2A-GFP | This study (XSB742) | CRISPR-cas9 vector for knocking out GADS |
| Plasmid | pHR-sfGFP | This study (XSB401) | Lentiviral vector with a superfold GFP tag |
| Plasmid | pHR-PLCγ1-sfGFP | This study (XSB434) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1ΔnSH2-sfGFP | This study (XSB838) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–549, 658–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1ΔSH3-sfGFP | This study (XSB840) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–790, 852–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 H380F-sfGFP | This study (LZB74) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291, H380F) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 R586K-sfGFP | This study (LZB75) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291, R586K) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 nSH2-cSH2-SH3-sfGFP | This study (XSB801) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 550–851) fused with an sfGFP tag |
| Plasmid | pHR-LAT-mCherry | This study (XSB376) | Lentiviral vector; human LAT (UniProt accession no.: O43561-2, aa 1–233) fused with an mCherry tag |
| Plasmid | pFastBac-Bv PLCγ1-SNAPf | This study (XSB490) | Protein expression; His10-TEV-Bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291) fusing SNAPf tag |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-cSH2-SH3 | This study (XSB406) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-cSH2 | This study (LZB14) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–790) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-SH3 | This study (LZB13) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–667, 757–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 cSH2-SH3 | This study (LZB16) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 658–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2 | This study (LZB15) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–667) |
| Plasmid | pGEX-ybbR-BV PLCγ1 cSH2 | This study (LZB17) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 658–790) |
| Plasmid | pET-His6-BV PLCγ1 SH3 | This study (LZB86) | Protein expression; His6-TEV-Bovine PLCg1 (UniProt accession no.: P08487, aa 791–851) |
| Plasmid | pET-MBP-His8-LAT 48-233-His6 | This study (XSB714) | Protein expression; MBP-TEV-His8-Human LAT (UniProt accession no.: O43561-2, aa 488–233)-TEV-His6 |
| Plasmid | pFastBac-His10-CD45 598-1304-SNAP | This study (XSB482) | Protein expression; His10-TEV-Human CD45 (UniProt accession no.: P08575, aa 598–1,304)-SNAP |
| Plasmid | pGEX-Grb2 | This study (XSB369) | Protein expression; GST-PreScission cleavage site-Human Grb2 (UniProt accession no.: P62993, aa 1–217) |
| Plasmid | pGEX-Sos1 1117-1319-Cys | This study (XSB370) | Protein expression; GST-PreScission cleavage site-Human Sos1 (UniProt accession no.: Q07889, aa 1,117–1,319)-Cys |
| Primer | pU6 F | This study (XSP130) | To verify plasmids for CRISPR knockout, 5′-GAGGGCCTATTTCCCATGATTCC-3′ |
| Primer | pHR F | This study (XSP79) | To verify plasmids for lentiviral infection, 5′-AGTCCTCCGACAGACTGAGT-3′ |
| Primer | pHR R | This study (XSP80) | To verify plasmids for lentiviral infection, 5′-CTTTCACAAATTTTGTAATCCAGAGGTTG-3′ |
| Primer | GST F | This study (XSP29) | To verify bacteria plasmids for recombinant protein production, 5′-ATGGCCTTTGCAGGGCTGG-3′ |
| Primer | PH F | This study (XSP5) | To verify baculoviral plasmids for recombinant protein production, 5′-AAATGATAACCATCTCGC-3′ |
| Primer | Fastback R | This study (XSP6) | To verify baculoviral plasmids for recombinant protein production, 5′-CAAGTAAAACCTCTACAAATGTG-3′ |
| Antibody | Anti-human CD3 | eBioscience | CD3 monoclonal antibody (OKT3), Cat#16-0037-85 |
| Antibody | Anti-human CD28 | eBioscience | Cat#16-0289-85 |
| Antibody | Anti-human pERK | Cell Signaling | Phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204), Cat#9101, Western blot (1:4,000) |
| Antibody | Anti-human PLCγ1 pY783 | Cell Signaling | Cat#2821, Western blot (1:3,000) |
| Antibody | Anti-human PLCγ1 | Cell Signaling | Cat#5690, Western blot (1:4,000) |
| Antibody | Anti-human pSLP76 | Abcam | Anti-SLP76 (phospho Y145), Cat#ab75829, Western blot (1:2,000) |
| Antibody | Anti-human LAT pY132 | Invitrogen | Cat#44-224, Western blot (1:2,000) |
| Antibody | Anti-human LAT pY171 | Cell Signaling | Cat#3581, Western blot (1:2,500) |
| Antibody | Anti-human LAT pY191 | Cell Signaling | Cat#3584, Western blot (1:2,500) |
| Antibody | Anti-human LAT | Millipore | Cat#05-770, Western blot (1:500) |
| Antibody | Anti-GAPDH | BioLegend | Cat#649202, Western blot (1:4,000) |
| Antibody | Anti-mouse IgG | Invitrogen | Goat anti-mouse IgG (H+L) secondary antibody, Cat#31430, Western blot (1:15,000) |
| Antibody | Anti-rabbit IgG | Invitrogen | Goat anti-rabbit IgG (H+L) secondary antibody, Cat#31460, Western blot (1:10,000) |
| Chemical | Vanadate | New England Biolabs | Sodium orthovanadate (Vanadate), Cat#P0758S |
| Reagent type . | Designation . | Source or reference . | Additional information . |
|---|---|---|---|
| Cell line | HEK293T | University of California, San Francisco, Cell Culture Facility | Lentivirus package |
| Cell line | Jurkat T cell E6.1 | University of California, San Francisco, Cell Culture Facility | Stable expression cell line construction |
| Cell line | PLCG1 null | This study (J74B) | PLCG1 knockout by CRISPR |
| Cell line | GRB2 null | This study (J81) | GRB2 knockout by CRISPR |
| Cell line | GADS null | This study (J82) | GADS knockout by CRISPR |
| Plasmid | pMD2.G | Vale Laboratory (XSB395) | Viral packaging, plasmid |
| Plasmid | poPAX | Vale Laboratory (XSB396) | Viral packaging, plasmid |
| Plasmid | pSpCas9(BB)-PLCG1 234F-2A-GFP | This study (XSB717) | CRISPR-cas9 vector for knocking out PLCG1 |
| Plasmid | pSpCas9(BB)-Grb2 42F-2A-GFP | This study (XSB740) | CRISPR-cas9 vector for knocking out GRB2 |
| Plasmid | pSpCas9(BB)-Gads 97F-2A-GFP | This study (XSB742) | CRISPR-cas9 vector for knocking out GADS |
| Plasmid | pHR-sfGFP | This study (XSB401) | Lentiviral vector with a superfold GFP tag |
| Plasmid | pHR-PLCγ1-sfGFP | This study (XSB434) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1ΔnSH2-sfGFP | This study (XSB838) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–549, 658–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1ΔSH3-sfGFP | This study (XSB840) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–790, 852–1,291) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 H380F-sfGFP | This study (LZB74) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291, H380F) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 R586K-sfGFP | This study (LZB75) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291, R586K) fused with an sfGFP tag |
| Plasmid | pHR-PLCγ1 nSH2-cSH2-SH3-sfGFP | This study (XSB801) | Lentiviral vector; bovine PLCγ1 (UniProt accession no.: P08487, aa 550–851) fused with an sfGFP tag |
| Plasmid | pHR-LAT-mCherry | This study (XSB376) | Lentiviral vector; human LAT (UniProt accession no.: O43561-2, aa 1–233) fused with an mCherry tag |
| Plasmid | pFastBac-Bv PLCγ1-SNAPf | This study (XSB490) | Protein expression; His10-TEV-Bovine PLCγ1 (UniProt accession no.: P08487, aa 1–1,291) fusing SNAPf tag |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-cSH2-SH3 | This study (XSB406) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-cSH2 | This study (LZB14) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–790) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2-SH3 | This study (LZB13) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–667, 757–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 cSH2-SH3 | This study (LZB16) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 658–851) |
| Plasmid | pGEX-ybbR-BV PLCγ1 nSH2 | This study (LZB15) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 550–667) |
| Plasmid | pGEX-ybbR-BV PLCγ1 cSH2 | This study (LZB17) | Protein expression; GST-PreScission cleavage site-ybbR tag-Bovine PLCγ1 (UniProt accession no.: P08487, aa 658–790) |
| Plasmid | pET-His6-BV PLCγ1 SH3 | This study (LZB86) | Protein expression; His6-TEV-Bovine PLCg1 (UniProt accession no.: P08487, aa 791–851) |
| Plasmid | pET-MBP-His8-LAT 48-233-His6 | This study (XSB714) | Protein expression; MBP-TEV-His8-Human LAT (UniProt accession no.: O43561-2, aa 488–233)-TEV-His6 |
| Plasmid | pFastBac-His10-CD45 598-1304-SNAP | This study (XSB482) | Protein expression; His10-TEV-Human CD45 (UniProt accession no.: P08575, aa 598–1,304)-SNAP |
| Plasmid | pGEX-Grb2 | This study (XSB369) | Protein expression; GST-PreScission cleavage site-Human Grb2 (UniProt accession no.: P62993, aa 1–217) |
| Plasmid | pGEX-Sos1 1117-1319-Cys | This study (XSB370) | Protein expression; GST-PreScission cleavage site-Human Sos1 (UniProt accession no.: Q07889, aa 1,117–1,319)-Cys |
| Primer | pU6 F | This study (XSP130) | To verify plasmids for CRISPR knockout, 5′-GAGGGCCTATTTCCCATGATTCC-3′ |
| Primer | pHR F | This study (XSP79) | To verify plasmids for lentiviral infection, 5′-AGTCCTCCGACAGACTGAGT-3′ |
| Primer | pHR R | This study (XSP80) | To verify plasmids for lentiviral infection, 5′-CTTTCACAAATTTTGTAATCCAGAGGTTG-3′ |
| Primer | GST F | This study (XSP29) | To verify bacteria plasmids for recombinant protein production, 5′-ATGGCCTTTGCAGGGCTGG-3′ |
| Primer | PH F | This study (XSP5) | To verify baculoviral plasmids for recombinant protein production, 5′-AAATGATAACCATCTCGC-3′ |
| Primer | Fastback R | This study (XSP6) | To verify baculoviral plasmids for recombinant protein production, 5′-CAAGTAAAACCTCTACAAATGTG-3′ |
| Antibody | Anti-human CD3 | eBioscience | CD3 monoclonal antibody (OKT3), Cat#16-0037-85 |
| Antibody | Anti-human CD28 | eBioscience | Cat#16-0289-85 |
| Antibody | Anti-human pERK | Cell Signaling | Phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204), Cat#9101, Western blot (1:4,000) |
| Antibody | Anti-human PLCγ1 pY783 | Cell Signaling | Cat#2821, Western blot (1:3,000) |
| Antibody | Anti-human PLCγ1 | Cell Signaling | Cat#5690, Western blot (1:4,000) |
| Antibody | Anti-human pSLP76 | Abcam | Anti-SLP76 (phospho Y145), Cat#ab75829, Western blot (1:2,000) |
| Antibody | Anti-human LAT pY132 | Invitrogen | Cat#44-224, Western blot (1:2,000) |
| Antibody | Anti-human LAT pY171 | Cell Signaling | Cat#3581, Western blot (1:2,500) |
| Antibody | Anti-human LAT pY191 | Cell Signaling | Cat#3584, Western blot (1:2,500) |
| Antibody | Anti-human LAT | Millipore | Cat#05-770, Western blot (1:500) |
| Antibody | Anti-GAPDH | BioLegend | Cat#649202, Western blot (1:4,000) |
| Antibody | Anti-mouse IgG | Invitrogen | Goat anti-mouse IgG (H+L) secondary antibody, Cat#31430, Western blot (1:15,000) |
| Antibody | Anti-rabbit IgG | Invitrogen | Goat anti-rabbit IgG (H+L) secondary antibody, Cat#31460, Western blot (1:10,000) |
| Chemical | Vanadate | New England Biolabs | Sodium orthovanadate (Vanadate), Cat#P0758S |