Ecl chromogenic substrate

Whole cell lysate was prepared in ice-cold 1X RIPA lysis buffer (Millipore SIGMA). The total protein lysate concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific) according to the protocols of manufacturers. Twenty-five micrograms of protein extracts were resolved on a 10 or 15% SDS polyacrylamide gel and electroblotted onto a nitrocellular membrane (Millipore). Membrane was blocked with 5% non-fat milk, then incubated with primary antibodies (Supplementary Table 3) at 4°C overnight. Horseradish peroxidase-conjugated secondary antibody (1:3000, Cell Signaling Technology) was used for chemiluminescent detection. Immunoreactivity signals were visualized using ECL chromogenic substrate (Bio-Rad Laboratories).

Cells were lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology, Jiangsu, China) supplemented with 1% protease inhibitor (Sigma-Aldrich, Saint Louis, MO, USA). Protein concentration was determined using a BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Jiangsu, China). Total protein (30-40 μg per sample) was resolved by SDS-PAGE using a 10% gel electrophoresis. Thereafter, protein content was transferred to polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) and then incubated with 5% nonfat milk in Tris-buffer saline containing 0.1% Tween 20 (TBST) for 1-2 hrs at room temperature. Next, the membrane was incubated overnight at 4°C with primary antibody against C1QTNF6 (ab36900, 1 : 1000 dilution, Abcam, USA) or GAPDH (AB0037, 1 : 5000 dilution, Abways, China). After washing with TBST, the membrane was then incubated with horseradish peroxidase- (HRP-) conjugated secondary antibodies (#7074, Cell Signaling Technology, USA). Respective protein signals were visualized by enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). GAPDH was used as a loading control.

Total protein was extracted from transfected and non-transfected cells using RIPA buffer (Beyotime, China) containing a protease inhibitor cocktail (Roche, USA). Protein concentration was quantified by a BCA protein assay kit (Beyotime, China). Lysates were combined with equal volumes of 2× Laemmli sample buffer. After 5 minutes of boiling, 15 μg of lysates were SDS-PAGEd and transferred to a 0.2 μm immune-BlotTM PVDF membrane (Cat. No. 162-017777; Bio-Rad Laboratories, CA, USA). 5% BSA (Cat. No. A-7888; Sigma Aldrich, MO, USA) in 0.1% Tween 20 blocked the membranes for 1 h. The primary antibodies for Western blotting were anti-PD-L1 (Cat. No. ab233482; Abcam) and anti-GAPDH (Cat. No. ab9485; Abcam). After the washing step, the membrane was incubated with secondary antibodies (goat anti-rabbit IgG H&L (HRP) (Cat No. ab6721; Abcam). Then, an ECL chromogenic substrate (BIO-RAD, USA) was applied to detect the signals. Densitometry of protein bands was done using Quantity One software to divide the percentage area under the curve of each band by its corresponding GAPDH band, and then calculated values were compared between them.