Zap70

Antibodies used for T cell stimulation included DimerX I recombinant HLA-A2 Ig (BD Biosciences), CD3 (clone OKT3, Biolegend), and CD28 (clone CD28.2, Biolegend). Western blot antibodies included PTPN22 (clone D6D1H, Cell Signaling Technology), Lck (Cell Signaling Technology), Zap-70 (Cell Signaling Technology), α-tubulin (clone TU-02, Santa Cruz), anti-rabbit Ig (Life Technologies) and anti-mouse Ig (LI-COR). For IP-FCM, the Akt capture antibody used was clone SKB1 (Merck Millipore), total Akt was detected using clone 53G (Cell Signaling Technology), and phospho Akt S473 was detected using clone D9E (Cell Signaling). Flow cytometry antibodies included CD69 (clone FN50, BD Biosciences), Il-2 (clone MQ1-17H12, Biolegend), pSrc family Y416 (Cell Signaling Technology), Lck pY505 (Cell Signaling Technology), Zap-70 pY493 (Cell Signaling Technology), Erk 1/2 pY204 (clone 197G2, Cell Signaling Technology), NFAT (clone D43B1, Cell Signaling Technology), cFos (clone 2G2, Novus Biologicals), an anti-rabbit secondary (Invitrogen), and an anti-mouse secondary (Life Technologies).

ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Akt (pan), phospho-Akt (S473), CD79b, CD79a, phospho-CD79a (Tyr182), SHP1, phospho-SHP1 (Tyr564), SHIP1, phospho-SHIP1 (Tyr1020), BTK, phospho-BTK(Tyr223), NF-kB p65, phospho–NF-kB p65 (ser536), IgM, Lyn, phospho-Lyn (Y507), LCK, CD86, and ZAP70 antibodies were from Cell Signaling Technology (Beverly, MA). Anti-SRC family (phospho-Y418)–phospho-Lyn (Y396), CD62L, and IgG antibodies were from Abcam (Cambridge, UK). Purified anti-human actin antibody was obtained from MP Biomedicals (Illkirch, France). Goat anti Rabbit IgG (H+L)–HRP conjugate and Goat anti Mouse IgG (H+L)–HRP conjugate, Goat F(ab′)2 anti-human IgM and Goat F(ab′)2 anti-human IgG were from Jackson Immunoresearch Laboratories (West Grove, PA). All antibodies utilized in the study were used in concentrations according to the manufacturer’s instructions. Ficoll-Paque PLUS from GE healthcare (Uppsala, Sweden), dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany), RPMI, fetal calf serum (FCS), Dulbecco’s phosphate-buffered saline (PBS), L-glutamine, and penicillin-streptomycin from Biological Industries (Beit-Haemek, Israel) were utilized for cell cultures.

Antibodies against HLA-A2 (BB7.2), HLA-ABC (W6/32), CD8a (RPA-T8), TNF (MAb11), and IFN-γ (4 S.B3) were purchased from eBioscience. Anti-CD3 (SK7), CD107a (H4A3), CD247-Alexa Fluor 488 (pY142) (K25-407.69), pCD3ζ (pY142) (K25-407.69), Erk1 (Cat# 610031), and PLC-γ1 (Cat# 610027) were from Becton Dickinson. Anti-pLAT (Tyr191) (Cat# 3584S), P-p44/42 MAPK (T202/Y204) (Cat# 4370L), pSrc family (pY416) (Cat# 6943T), non-pSrc family (pY416) (Cat# 2102S), pSHP-1 (Cat# 8849S), pZap70 (pY319) (Cat# 2717S), Zap70 (Cat# 2709), pPLC-γ1 (pY783) (Cat# 2821S), α-tubulin (Cat# 3873S), GAPDH (Cat# 2118S) antibodies were from Cell Signaling Technologies. Anti-LAT (11B.12) and β-2-microglobulin (2M2) antibodies were from Biolegend. Mouse anti-human HLA-A2-E183-91 and mouse antihuman HLA-A2-C18-27 primary antibodies were produced as described^{18 (link)}. F(ab’)2-goat anti-rabbit IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 488 (Cat# A-11070), and goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor 647 (Cat# A-21237) were purchased from Thermo Fisher Scientific.

Purified T cells were serum starved overnight in complete medium supplemented with 0.5% FCS, and subsequently activated on anti-CD3 antibodies (1 μg/ml) coated plates in the presence of anti-CD28 antibodies (1 μg/ml) with or without SRA-ECD protein (10 μg/ml). Activated cells were lysed using RIPA buffer (50 mM Tris, 150 mM NaCl, and 1% Nonidet P-40, pH 7.4) and protein lysates were subjected to western blot analysis with antibodies against phospho-ZAP70, ZAP-70 (Cell Signaling Technology, Danvers, MA, USA) or GAPDH (Thermo scientific Inc., Waltham, MA, USA).