Anti pstat1 tyr701 58d6

Antibodies used in the experiments with mouse cells were obtained from Biolegend unless otherwise stated: anti-CD3 (17A2); anti-CD4 (GK1.5); anti-CD8 (53-6.7); anti-B220 (RA3-6B2); anti-CD25 (PC61.5); anti-CD44 (IM7); anti-CD45 (30-F11); anti-CD62L (MEL-14); anti-CD45.1(A20); anti-CD45.2 (104); anti-NKG2D (CX5); anti-PD-1 (29F.1A12); anti-TIM-3 (RMT3-23); anti-CD200 (OX-90); anti-EpCAM (G8.8); anti-MHC I (36-7-5); anti-MHC II (M5/114.15.2); anti-LAG-3 (C9B7W; Thermo Fisher Scientific); anti-pan NK cells (DX5; Thermo fisher); anti-Foxp3 (FJK-16s; Thermofisher); IL-17 (TC11-18H10.1); anti-TNF-α (TN3-19.12); anti-IFN-γ (XMG1.2); anti-Eomes (Dan11mag; Thermo fisher); anti-TOX (REA473; Miltenyi Biotec), anti-pStat1 (Tyr701) (58D6; Cell Signaling); anti-pStat5 (Tyr694) (C71E5; Cell Signaling); anti-Actin (SCBT). The following mAbs raised against human antigens were all purchased from Biolegend: CD3 (OKT3); CD4 (OKT4); CD8 (SK1); NKG2D (1D11); IL-17 (BL168); IFN-γ (B27). Viable cell populations were gated based on forward and side scatters and by fixable blue (Thermo Fisher Scientific) staining. Samples were collected on an LSR II and analyzed with FlowJo software (Tree Star).

Sub-confluent cells were lysed in Laemmli buffer (LB) and 45 µg of total proteins were subjected to SDS-PAGE and western blotting following standard methods. Protein detection was performed by using the following primary antibodies, diluted as indicated: anti-human MET (3D4, 1:3000, Invitrogen Corp., Camarillo, CA), anti-pMET Tyr^1234/1235 (D26, 1:1000), anti-JAK1 (6G4, 1:1000), anti-pJAK1 Tyr^1022/1023 (1:1000), anti-JAK2 (D2E12, 1:1000), anti-pJAK2 Tyr^1007/1008 (1:1000) anti-STAT1 (1:1000), anti-pSTAT1 Tyr⁷⁰¹ (58D6, 1:1000), anti-PD-L1 (E1L3N, 1:1000), anti-GAPDH (D4C6R, 1:1000) (all from Cell Signaling Technology, Beverly, MA), anti-IFNGR1 (EPR7866, 1:1000, Abcam, Cambridge, UK) and anti-Vinculin (hVIN-1, 1:1000, Sigma Life Sciences). Secondary HRP-conjugated goat anti-mouse IgG (1:20,000) or anti-rabbit IgG (1:20,000) (from Jackson ImmunoResearch, Cambridge, UK) and ECL System (Promega, Madison, WI) were used for protein detection.