Hrp conjugated goat anti mouse or anti rabbit igg antibody

Total protein was extracted from HCC cells, and 20 μg of isolated proteins were separated by 10% SDS-PAGE, transferred onto a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA), and probed with the respective primary antibodies (1:1000, Cell Signaling, Danvers, MA, USA) for overnight. Subsequently, the membranes were incubated with the HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (ZSGB-BIO, China). Protein bands were visualized using an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK).

Total protein was harvested from GC cells using RIPA buffer (Wolsen, China) after 48 h transfection and 20 μg of isolated protein lysates were separated by 10% SDS-PAGE and transferred to PVDF membrane (Millipore, USA). The membranes were probed with the following primary antibodies: IGF-1R, AKT, phospho-AKT (Ser473), CCND1, CDK4/6, CDK2, Bcl-2 and Bax (Cell Signaling Technology, diluted 1/1,000) overnight at 4 °C. The expression levels of above proteins were standardized to human β-actin using a mouse mAb anti-β-actin antibody. Then, the membranes were incubated with the HRP-conjugated goat anti-mouse or anti-rabbit IgG antibody (ZSGB-BIO, China). The blots were scanned, and the band density was measured using the Quantity One imaging software.