Chemiluminescence detection system

The cells were lysed in chilled RIPA lysis buffer on ice, and then centrifuged at 12,000 × g for 20 min at 4°C and collected the supernatants. Protein concentrations were assessed with a BCA Protein Assay kit (Bio-Rad Laboratories, Inc.). Protein lysate was separated by 10% SDS-PAGE, followed by being electroblotted onto a PVDF membrane (DuPont, Boston, MA, USA), which had been blocked with 5% non-fat milk in TBST for 1 h at room temperature. Next, the membrane was incubated with the primary antibodies: anti-GOLM1 antibody (1:300, cat. no. HPA010638; Atlas Antibodies, Stockholm, Sweden) and anti-GAPDH antibody (1:3,000; cat. no. ab9485) at 4°C overnight, and then rinsed the membrane and incubated it with anti-rabbit IgG (1:4,000; cat. no. ab191866) (both from Abcam, Cambridge, UK) for 2 h at room temperature. Chemiluminescence detection system (Beyotime Institute of Biotechnology, Shanghai, China) was used to detect the results and the protein expression was normalized to GAPDH.

Radioimmunoprecipitation assay buffer (Beyotime, Shanghai, China) was used to isolate total proteins according to the manufacturer’s instruction. Protein samples of 40 μg were loaded onto the sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinyl fluoride membrane. After blocking with 5% non-fat milk for 1.5 hours at room temperature, the membranes were incubated with primary antibodies for overnight at 4°C. The primary antibodies used were as follows: anti-CBX5 (#2616; Cell Signaling Technology, Danvers, Massachusetts) and anti-GAPDH (#5174; Cell Signaling Technology). After washing with Tris-buffered saline Tween, membranes were incubated with the horseradish peroxidase–linked goat antirabbit secondary antibody (#7074; Cell Signaling Technology) for 2 hours at room temperature. Band signals were developed by a chemiluminescence detection system (Beyotime).