Pd l1

Briefly, cells were lysed with RIPA buffer (keyGEN, China) containing protease inhibitors. 20 μg of protein per sample was loaded, run on 10% SDS–polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. After being blocked with 5% bovine serum albumin, membranes were incubated with primary antibodies including PD-L1 (66248, Proteintech), JAK1(3344T, Cell signaling technology), STAT6 (51073, Proteintech) GAPDH (ab8245, Abcam) at 4°C overnight, 1 h of incubation with secondary antibodies and detected by FlourChemE system (Protein Simple).

For FACS analysis, cells were stained using an indirect staining method. Cells were collected from the flasks (with or without treatment), and were fixed using ice-chilled 70% ethanol at 4 °C. The cells were then washed and incubated with primary antibodies (as mono-staining) overnight at 4 °C at a concentration of 1 µg for HLA-G (Epigentek, USA) and 1.25 µg for PD-L1 (Proteintech, USA) per 100,000 cells. In parallel, the cells were stained with equivalent amount of isotype antibodies (Figure S4) i.e., rabbit polyclonal IgG and mouse monoclone IgG1 antibody (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). On the following day, the cells were then washed and incubated with secondary antibodies (anti-rabbit AF488 and anti-mouse AF546; Invitrogen) for 2 h. To stain the cells in mitosis, we used MPM2 antibody (Anti-phospho-Ser/Thr-Pro, Cy5 conjugate; Sigma-Aldrich, Saint Quentin Fallavier, France) at concentration of 2 µg per 100,000 cells. The cells were analyzed using 3-Laser Flow cytometer “Gallios” by Beckmann Coulter and the results were analyzed using “Kaluza Analysis 2.1” software. Cell cycle was recorded through DAPI, using Michel H. Fox algorithm (built-in Software).

For western-blot analysis, 50 μg of protein from in vivo tissue lysates were resolved by gel electrophoresis and electrotransferred to polyvinylidene difluoride membranes by semi-dry transfer (Biorad Trans-blot SD, Hercules, CA, USA). Blots were blocked with 4% dry powder milk or BSA for 1 h and then incubated with primary antibodies for p-EGFR (Y845), p-EGFR (Y1068), p-EGFR (Y1173), total EGFR, p-Src (Y418), total Src, pSTAT3 (Y705), total STAT3, pSTAT5 (Y694/Y699), total STAT5, COX-2, Cyclin D1, and Cyclin D2, IFN-γ, TLR-4, phospho-p38, IFN-β. These antibodies were all acquired from Santa Cruz Biotech (Santa Cruz, CA, USA); PD-L1 was acquired from Proteintech (Rosemont, IL). Blots were incubated with primary antibodies at 4°C overnight and after washing were incubated for 1 h at room temperature with respective secondary antibodies conjugated to peroxidase (Santa Cruz Biotechnology, Dallas, TX, USA). Blots were then developed with an ECL detection system. Densitometric analysis was performed using ImageJ 1.x software [42 (link)].

The LMP1 antibody was purchased from DAKO (Cambridge, MA, USA) and the β-actin antibody was from Sigma-Aldrich (St. Louis, MO, USA). The cleaved-PARP and cleaved-Caspase 3 antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Ubiquitin and PD-L1 antibodies were purchased from Proteintech (Chicago, IL, USA). The Flag-Tag monoclonal antibody and Myc-Tag monoclonal antibody antibodies were from Immunoway Biotechnology Company (Plano, Texas, USA). The PGC-1α antibody was obtained from Novus Biologicals (Littleton, CO, USA) and the PRMT1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The dimethyl arginine antibody was obtained from ImmuneChem Pharmaceuticals Inc (Burnaby British Columbia, Canada). Cycloheximide was obtained from Sigma-Aldrich (Saint Louis, MO, USA), and MG132 was purchased from MedChemExpress (Shanghai, China).

FFPE tissue from the PUMCH_Uro cohort was used for the IHC assay as previously reported (18 (link)). Paraffin sections were first incubated with primary antibodies against CD8A (Proteintech, China) and PD-L1 (Proteintech, China) and then a goat peroxidase-conjugated secondary antibody (Proteintech, China). After washing, the sections were stained with DAB and hematoxylin for signal visualization.

Cellular proteins were extracted with protein extraction reagent (Beyotime Biotechnology) and quantified by BCA protein assay (Beyotime Biotechnology). A total of 12 μg of protein per sample was added to SDS-PAGE gels for electrophoresis (100 V, 2 h), followed by constant flow membrane transfer (ice bath, 210 mA, 2 h). The transferred polyvinylidene fluoride (PVDF) membranes were blocked with 5% BSA-containing Tris-buffered saline with Tween (TBST) for 2 h at room temperature, and incubated with primary antibodies overnight at 4°C. Then, membranes were washed three times with TBST and incubated with secondary antibody at room temperature for 2 h. Membranes were examined with a gel imager (ECL, Millipore, USA). Antibodies of Vimentin, E-Cadherin, smad2, ERK, p-ERK, CREB, and GAPDH were purchased from Cell Signaling Technology (CST); YAP1, p-YAP1, and PD-L1 were purchased from Proteintech. All primary antibodies were used at 1:1,000 dilution. Secondary antibodies were purchased from Beyotime Biotechnology (1:2,000 dilution).