Proteins were extracted from cells with RIPA buffer complemented with phenylmethanesulfonyl fluoride (PMSF, Beyotime). Protein extracts were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Immunoblots were blocked with 5% bovine serum albumin (BSA) in TBS/Tween 20 and incubated with primary antibodies overnight at 4 °C. The bands were visualized with the FluorChem E System (Protein Simple, USA).
The following antibodies diluted in 5% BSA were used: anti-GAPDH (sc-47724, 1:500), anti-p62 (sc-28359, 1:1000), and anti-Beclin-1 (sc-48341, 1:1000) were purchased from Santa Cruz Biotechnology. Anti-LC3B (3868 T, 1:2000) and anti-PIK3C3 (4263S, 1:2000) were purchased from Cell signal technology (CST). Anti-Cezanne (16605-1-AP, 1:1000), anti-POLR2A (20655-1-AP, 1:1000), anti-PIK3C2A (22028-1-AP, 1:1000), and anti-Flag (20543-1-AP, 1:1000) were purchased from Proteintech (Wuhan, Hubei, China). Anti-Myc (TA150121, 1:1000), anti-HA (TA180128, 1:1000), and anti-GST (TA150101, 1:1000) were purchased from Origene (Wuxi, Jiangsu, China).
The following antibodies diluted in 5% BSA were used: anti-GAPDH (sc-47724, 1:500), anti-p62 (sc-28359, 1:1000), and anti-Beclin-1 (sc-48341, 1:1000) were purchased from Santa Cruz Biotechnology. Anti-LC3B (3868 T, 1:2000) and anti-PIK3C3 (4263S, 1:2000) were purchased from Cell signal technology (CST). Anti-Cezanne (16605-1-AP, 1:1000), anti-POLR2A (20655-1-AP, 1:1000), anti-PIK3C2A (22028-1-AP, 1:1000), and anti-Flag (20543-1-AP, 1:1000) were purchased from Proteintech (Wuhan, Hubei, China). Anti-Myc (TA150121, 1:1000), anti-HA (TA180128, 1:1000), and anti-GST (TA150101, 1:1000) were purchased from Origene (Wuxi, Jiangsu, China).