Gapdh 6c5

About 30 μg proteins were loaded into 10% SDS/PAGE gel and then transferred onto a PVDF membrane. After incubated with the primary antibody in a 4°C condition for overnight and secondary antibody at room temperature for 1–2 h, the membrane was visualized with ECL system. The SLTRK4 (ab67315), PCDH7 (ab139274) and GAPDH (6C5) antibodies were purchased from Abcam company. UGT2A3 (PA5-48900) antibody was purchased from Thermo Fisher Scientific company.

Affinity-purified rabbit antibodies against myosin VI, TAX1BP1 and optineurin were generated as previously described (Buss et al., 1998 (link); Morriswood et al., 2007 (link); Sahlender et al., 2005 (link)). Commercial antibodies against the following proteins were used: Cx43 [C6219; 1:200 immunofluorescence (IF), 1:2000 western blotting (WB)] and actin (A2066; 1:5000 WB) polyclonal antibodies (Sigma), Cx43 (05-763; 1:200 IF) monoclonal antibody (Millipore), GAPDH (6C5; 1:1000 WB) and clathrin (X22; 1:100 IF) monoclonal antibodies (Abcam), Dab2 (H-110; 1:100 IF) and GIPC1 (N-19; 1:100 IF) polyclonal antibodies (Santa Cruz Biotechnology) and pan-cadherin (4068; 1:1000 WB) polyclonal antibody (Cell Signaling). Phalloidin–Alexa-Fluor-647, Calcein AM, WGA–647 and Hoechst 33342 were purchased from ThermoFisher. Bafilomycin A1, brefeldin A and biotin were purchased from Sigma-Aldrich. JF₆₄₆-HaloTag ligand was a kind gift from Joel Slaughter and Luke Lavis (Janelia Farm, HHMI, Ashburn, VA) (Grimm et al., 2015 (link)).

Cells were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer (5 mm EDTA, 150 mm NaCl, 10 nm Tris-HCl, pH 7.2, 0.1% SDS, 0.1% Triton X-100, and 1% sodium deoxycholate) containing protease mixture inhibitors P8340, P5726, and P0044 (Sigma). Protein concentration was determined by bicinchoninic acid assay (BCA) (Thermo Scientific) according to the manufacturer's protocol, using BSA as standards. Protein samples were denatured and resolved on SDS-PAGE gels using a Bio-Rad PowerPac HC and transferred onto PVDF membranes (Millipore). Membranes were probed overnight at 4 °C (1:1000) for the following primary antibodies to PD-L1 (E1L3N), SOCS1 (A156), STAT1 (9172), P-STAT1 Tyr-701 (D4A7), STAT3 (9132), and P-STAT3 Tyr-705 (D3A7) all from Cell Signaling, and for 1 h at room temperature for GAPDH (6C5) and β-actin (ab6276), both from Abcam. Membranes were further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and visualized with ECL (GE Healthcare). Band intensity was quantified using ImageJ version 1.50e (NIH, Bethesda, MD).