Western Blot Analysis of Protein Expression

Total proteins were harvested, and western blots were performed as described previously (23 (link)). Briefly, proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. After blocking with 5% non-fat milk at 24°C for 1 h, the membranes were incubated with primary antibodies targeting HA-Tag (diluted 1:1000, Cell Signaling Technology) and glyceraldehyde 3-phosphate dehydrogenase (diluted 1:1000, Proteintech) at 4°C overnight. After that, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies (dilution 1:5000, Cell Signaling Technology) for 2 h at 25°C. Proteins were detected using a Bio-Rad Imaging System.