Ccnd1

Cells were lysed in RIPA buffer (Beyotime, Shanghai, China) with Protease Inhibitor Cocktail Tablets (Roche, Switzerland). The total protein concentrations of the lysates were determined using a protein assay kit (Bio-Rad, USA). Equal amounts of protein were denatured with loading buffer (Beyotime, Shanghai, China) at 100°C for 10 min, then loaded in 12% SDS-PAGE for electrophoresis, and transferred to a methanol-activated polyvinylidene fluoride membrane (Millipore, USA). The membrane was blocked in tris-buffered saline (TBS) containing 5% bovine serum albumin (MP Biomedical, USA) for 2h at room temperature. Primary antibodies were incubated overnight at 4°C. The primary antibodies include: RBL1 (1:1000, Proteintech, China), CCND1, CCNF (1:1000, Affinity Biosciences, USA) and GAPDH (1:1000, ZSGB-BIO, Beijing, China). After washing with TBS, the membrane was incubated with the appropriate horseradish peroxidase (HRP)-labeled secondary antibody for 1 h at room temperature. Secondary antibody conjugated with HRP is Goat-Anti Rabbit IgG (1:5000, ZSGB-BIO, Beijing, China). The relative protein level is quantified using Image J software.

Protein samples were extracted from glioma cell lines/glioma tissues. Protein samples were separated on 12.5% SDS-PAGE gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated with the primary antibody at 4 °C overnight. The next day, the membranes were incubated with fluorescent dye-conjugated secondary antibodies at room temperature for 2 h. The membranes were observed using a ChemiDoc XRS + Imaging System (Bio-Rad, USA). The following primary antibodies were utilized in the present study: P62 (18420-1-AP, Proteintech); MAP1LC3B (L7543, Sigma); CCND1 (AF0931, Affinity); CCNB1 (DF6786, Affinity); HAS3 (DF13055, Affinity); CD44 (A0340, Abclonal); and β-actin (TA-09, ZSGB-BIO).