Cyclin d1

Total protein was obtained from cell samples with the RIPA buffer (Beyotime, China) supplemented with protease inhibitor cocktail tablet (Roche, Switzerland). Western blot was carried out with rabbit antibodies targeting IGF2BP2 (1:5000, Abcam, UK), cyclinD1 (1:1000, Abways, china), AKT (1:1000, Abways, china), p-AKT (1:1000, Abways, china), and GAPDH (1:5000, ZENbio, China) antibodies. HRP-conjugated IgG (1:5000, Beyotime, China) was utilized as secondary antibodies. The ECL-Plus reagent (Millipore, USA) was utilized for development. GAPDH was used for normalization.

The transfected cells were lysed with RIPA lysate for 30 min to extract the total protein. Then total protein was divided by electrophoresis in SDS-PAGE system for 120 V, 2 h. Polyvinylidene fluoride (PVDF) membrane as the carrier and protein transferred for 400 mA, 1 h. The following antibodies were used: LC3B (Proteintech: 14,600–1-AP, 1:1000), p62 (Abmart: T59081, 1:1000), tubulin (Abmart: M20005, 1:1000), Caspase 3 (Abmart: T40044, 1:1000), bcl-2 (Abmart: T40056, 1:1000), CDK1 (Abways: CY5176, 1:1000), cyclin B1 (Abways: CY5378, 1:1000), cyclin D1 (Abways: CY5404, 1:1000), RAB11A (Abways: CY5301, 1:1000).

Proteins were isolated by RIPA buffer (Vazyme), and Detergent Compatible Bradford Protein Quantification Kit (Vazyme) was used to determine the concentration of proteins. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and then the proteins were transferred onto the polyvinylidene difluoride (PVDF) membranes (Vazyme). After being blocked with 5% skimmed milk (Vazyme) and washed by phosphate-buffered saline (PBS), the membranes were incubated with corresponding primary antibodies: MUC19 (1:1000 dilution, ab212621, Abcam, Cambridge, UK), GAPDH (1:2000, ab37168, Abcam), PCNA (1:3000, Abways Technology, Inc., Shanghai, China), cyclin D1 (1:1000, Abways Technology), and antibodies against hexokinase II (HK2) (ab227198, 1:5000, 102 kDa) overnight at 4 °C. Then the membranes were incubated with the secondary antibody (1:3000, ab205718, Abcam) for 3 h. The blots were analyzed by the ChemiDoc™ MP Imaging System (Bio-Rad, Richmond, CA, USA) after being incubated with ECL kit (Vazyme).