Pstat5

Fluorochrome-conjugated antibodies against CD3, CD8, CD44, CD62L, CD69, Tim-3, PD-1, CD90.1, CD45.1, Granzyme B, Tbet, Bcl-2, IFN-γ, IL-2 (Biolegend), NKG2D, CD25, TNF-α, KLRG1 (eBioscience), pSTAT5 (Invitrogen), CD127 (BD Bioscience) and pS6 (Cell Signaling Technologies) were used. Cell surface staining, intracellular staining, and flow cytometry analysis was performed as previously described [28 (link)]. Staining of phosphorylated proteins was performed following eBioscience protocol with methanol fixation and permeabilization. For analyses, cells were gated on live cells using Zombie Aqua exclusion dye (Biolegend). pMel cells were distinguished from endogenous cells by gating on the congenic marker CD90.1 and in vivo CTL assay analyses were performed after gating on the congenic marker CD45.1. When methanol was used, live cells were defined based on size.

Antibodies used in these studies were purchased from the following vendors: ERK1/2 (#9102), pERK1/2 (#9101), AKT (#9272), pAKT S473 (#9271), pAKT S473 (#3787S, for immunohistochemical analysis) from Cell Signaling Technology (Danvers, MA, USA); pSTAT5 (#71-6900) from Invitrogen (Grand Island, NY, USA); ERα (#sc-542), PRLR (#sc-20992), STAT5 (#sc-835x) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); eGFP (#AB6658) from AbCam (Cambridge, MA, USA); PR (#A0098) from Dako (Carpinteria, CA, USA); biotinylated goat anti-rabbit (#BA-100) from Vector Labs (Burlingame, CA, USA); pan-actin (#125-ACT) from Phosphosolutions (Aurora, CO, USA); APC-conjugated CD31 (#551262) and CD45 (#559864) from BD Biosystems (San Jose, CA, USA). Avidin-biotin complex (ABC) (#PK-4000) and ImmPACT DAB (#SK-4105) were purchased from Vector Labs (Burlingame, CA, USA). All other reagents were obtained from Fisher Scientific or Sigma-Aldrich.

Total protein was obtained from cells or tissues using a lysis buffer (Cell Signaling Technology, California, USA), and protein levels were quantified using the BCA kit (Shanghai Ze Ye Biotechnology Co., Ltd, Shanghai, China). Subsequently, approximately 30 μg of protein was loaded and separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride membrane (Millipore, Massachusetts, USA), followed by incubation with 5% bovine serum albumin and incubation with the primary antibodies against JAK1 (1:800, Invitrogen, Massachusetts, USA), p-JAK1 (1:800, Invitrogen, Massachusetts, USA), JAK2 (1:800, Invitrogen, Massachusetts, USA), p-JAK1 (1:800, Invitrogen, Massachusetts, USA), STAT3 (1:800, Invitrogen, Massachusetts, USA), p-STAT3 (1:800, Invitrogen, Massachusetts, USA), STAT5 (1:800, Invitrogen, Massachusetts, USA), p-STAT5 (1:800, Invitrogen, Massachusetts, USA), and GAPDH (1:800, Invitrogen, Massachusetts, USA). Subsequently, the membranes were incubated with a secondary antibody (1:2000; Invitrogen, Massachusetts, USA). Finally, enhanced chemiluminescence (Invitrogen, Massachusetts, USA) was used for the visualization of bands, followed by quantification using the ImageJ software (National Institutes of Health, USA) [19 (link)].