Bcl 10c4

For intracellular cytokine staining (ICS), cells stimulated with indicated stimuli in the presence of GolgiStop (BD Biosciences) were stained for cell-surface markers, fixed and permeabilized using Cytofix/Cytoperm buffer (BD Biosciences) and then stained with fluorochrome-conjugated Abs to IFN-γ (XMG1.2; eBioscience; 1:300) and TNF-α (MP6-XT22; BD Biosciences; 1:300) using Perm/Wash buffer (BD Biosciences). The same ICS protocol was used for analysing Bcl-2 and CD107a expression with fluorochrome-conjugated Abs to Bcl-2 (BCL/10C4; Biolegend; 1:200) and CD107a (1D4B; eBioscience; 1:300). Intracellular staining for T-bet and Eomes expression was performed with Foxp3 Staining Buffer Set (eBioscience) according to the manufacturer's instructions using fluorochrome-conjugated Abs to T-bet (4B10; 1:200) and Eomes (Dan11mag; all from eBioscience; 1:200). For intracellular staining for p-ERK, B6 SP cells treated with indicated stimuli were fixed with 2% paraformaldehyde at RT for 15 min, followed by permeabilization with ice-cold 90% methanol for 20 min on ice. After a washing step, cells were blocked with PBS containing 2% FBS and incubated with fluorochrome-conjugated Ab to p-ERK (Thr202/Tyr204; D13.14.4E; Cell Signaling Technology; 1:100), followed by repeated washes and continued incubation for 15 min on ice with fluorochrome-conjugated Abs to cell-surface markers.

The western blotting assay was performed by well-established protocols as previously described (19 (link)). Primary antibodies used in this study were anti-ABL1 antibody (1:500, ab85947, Abcam, Cambridge, MA), anti-Bcl-2 antibody (1:300, BCL/10C4, Biolegend, San Diego, CA), Anti-Bcl-xl antibody (1:500, sc-136207, Santa Cruz Biotechnology, Dallas, TX), anti-Bax antibody (1:300, 2D2, Biolegend), anti-β-actin antibody (1:500, 2F1-1, Biolegend), anti-GAPDH antibody (1:500, FF26A/F9, Biolegend), anti-p27 antibody (1:300, sc-56338, Santa Cruz Biotechnology), anti-cyclin-D1 antibody (1:500, sc-8396, Santa Cruz Biotechnology), anti-IRS1 antibody (1:500, ab52167, Abcam), anti-AKT2 antibody (1:500, ab175354, Abcam), anti-PPP3CA antibody (1:10000, ab52761, Abcam), anti-TGFβ1 antibody (1:100, ab92486, Abcam), anti-MAP2K2 antibody (1:500, sc-81473, Santa Cruz Biotechnology), anti-PI3K-p11a antibody (1:1000, ab151549, Abcam). Secondary antibodies used were: anti-mouse IgG HRP-conjugated secondary antibody (1:5000, sc-516102, Santa Cruz Biotechnology), and anti-rabbit IgG HRP-conjugated secondary antibody (1:5000, sc-2357, Santa Cruz Biotechnology).

The following antibodies were used for the flow cytometry analysis and cell sorting: CD4 (1:400, RM4-5, #553051), CD8 (1:400, 53-6.7, #553033), CD44 (1:400, IM7, #553134), CD25 (1:200, PC61, #562606), IL-2 (1:200, JES6-5H4, #554428), IL-4 (1:200, 11B11, #554435), IL-10 (1:200, JES5-16E3, #554467), IL-17 (1:200, TC11-18H10, #560184), GM-CSF (1:200, MP1-22E9, #564747), PD-1 (1:200, J43, #562584), CTLA-4 (1:200, UC10-4F10-11, #553720), and GITR (1:200, DTA-1, #558140) antibodies were purchased from BD Biosciences. Ki67 (1:100, anti-human, clone B56, #556027), CD103 (1:100, M290, #557495), and ICOS (1:200, 7E.17G9, #552146) antibodies were purchased from BD Pharmingen. IFN-γ (1:200, XMG1.2, #25-7311-41), Foxp3 (1:200, FJK-16s, #12-5773-82), LAG3 (1:200, C9B7W, #17-2231-82), Tim3 (1:200, RMT3-23, #12-5870-82), and KLRG1 (1:200, 2F1, #25-5893-82) antibodies were purchased from eBioscience. TIGIT (1:200, 1G9, #142103) and Bcl2 (1:100, BCL/10C4, #633503) antibodies were purchased from Biolegend. NGFR (1:800, NGFR5, #MS-394-B1) and Live/Dead cell stain kit (#L34955) were purchased from Thermo Fisher Scientific.