Anti β actin rabbit polyclonal antibody

The total cellular proteins from each group were extracted using RIPA lysis buffer with 1% phenylmethanesulfonyl fluoride (PMSF). Then, equal amounts (20 μg) of protein determined by BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) were separated using 10% SDS-PAGE gels. The proteins were then transferred to PVDF membranes (0.45 mm, Solarbio, Beijing, China). The membranes were blocked with 5% nonfat milk for 1 h at room temperature and then incubated with anti-IGF2BP3, Cyclin D1 (1:1000, Proteintech Group. Inc) rabbit polyclonal antibodies at 4 °C for 12 h. anti-β-actin rabbit polyclonal antibody (1:4000, Proteintech Group. Inc) was used as loading controls and normalization. The secondary antibodies were anti-mouse or anti-rabbit antibody and conjugated to horseradish peroxidase (HRP) (1:4000, Proteintech Group. Inc). The secondary antibodies were used at a 1:4000 dilution and were incubated for approximately 1 h at room temperature. The bands were visualized with ECL reagents (Thermo Fisher Scientific) and developed by Omega Lum G (Aplegen, USA).

Total cellular proteins were extracted from each group using a cell lysis solution (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts (20 μg) of protein were determined using a BCA protein assay kit (Thermo Fisher Scientific) and separated on 10% SDS-PAGE gels. Proteins were then transferred to PVDF membranes (0.45 um, Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked for 2 h at 37 °C with 5% non-fat milk in Tris Buffered saline with Tween 20 (TBST) and then incubated with anti-IGF2BP3 (1:1000, Cat No.14642-1-AP, Proteintech Group. Inc.), METTL3 (1:1000, Cat No. 15073-1-AP, Proteintech Group. Inc), and N-myc (1:1000, Cell Signaling Technology, Boston, MA, USA) rabbit polyclonal antibodies at 4 °C for 12 h. Anti-β-actin rabbit polyclonal antibody (1:4000, Proteintech Group. Inc.) was used as the loading control and normalization. The secondary antibodies were anti-rabbit IgG conjugated to horseradish peroxidase (HRP) (Proteintech Group. Inc). The secondary antibodies were used at a 1:4000 dilution and were incubated for 1 h at 37 °C. The bands were visualized with ECL reagents (Thermo Fisher Scientific, Waltham, MA, USA) and developed using Tanon 5200 (Tanon, Shanghai, China).