Anti socs2

Total cell lysate was prepared with radioimmunoprecipitation assay (RIPA) buffer containing Protease Inhibitor Cocktail, and we measured protein concentrations by bicinchoninic acid protein assay reagent (Bio-Rad) and resolved extracts using sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels. Membranes were blocked for 2 h at room temperature in TBS-Tween-20 containing 5% nonfat dried milk (Bio-Rad), washed, and then incubated with primary antibodies at 4 °C overnight. The following primary antibodies were used: anti-AURKA (1:1000, Cell Signaling Technology), anti-caspase-3 (1:1000, Cell Signaling Technology), anti-PARP (1:1000, Cell Signaling Technology), anti-cleaved-caspase-3 (1:1000, Cell Signaling Technology), anti-cleaved-PARP (1:1000, Cell Signaling Technology), anti-pH2AX (1:1000, Cell Signaling Technology), anti-SOCS2 (1:1000, Cell Signaling Technology), anti-flag (1:1000, Cell Signaling Technology), and anti-GAPDH (1:1000, Cell Signaling Technology). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology), and resulting signal was detected using enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA).

Antibodies for cell sorting include anti-B220, anti-CD2, anti-CD3, anti-CD5, anti-CD8, anti-Gr-1, anti-CD41, anti-Ter119, anti-CD150, anti-CD48, anti-Kit and anti-Sca1 (Table S2). All antibodies were purchased from BioLegend. Anti-APC conjugated to paramagnetic microbeads were from Miltenyi Biotec. Antibodies for western blotting were anti-pStat5 (Y694), anti-Stat5 (4H1), anti-β-actin (8H10D10), anti-pJak2 (Tyr221), anti-Jak2, anti-pJAK1 (Tyr1034/1035) and anti-SOCS2 (Table S3), all purchased from Cell Signaling Technology. Anti-GAPDH was from Santa Cruz.

Western blot was performed according to a standard method, as described previously [28 (link)]. The following primary antibodies were used: anti–SOCS2, anti–SOCS4, anti–SOCS6, anti–PTPN4, anti–PTPN11, anti–STAT3 and a-tubulin (Cell Signaling Technology); p84 was purchased from Abcam (Cambridge, USA). Nuclear extracts were prepared using the Nuclear Extraction Kit (Active Motif), according to the manufacturer’s instructions. All protein expression levels in this manuscript were quantified by densitometry using Quantity One Software, and normalized to the corresponding expression levels of α-tubulin respectively. The sample 1 was used as a standard.