Cdk4 6

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.

Immunohistochemistry (IHC) was performed using specific antibody in paired primary HNSCC and pre-neoplastic tissue specimens as described above [4 (link)]. MDA-9/Syntenin antibody was obtained from Abnova Corporation (dilution 1:200). The EGFR, CCND1, CDK4/6, CTNNB1, PI3K, STAT3, EF1-alpha and Beta-actin antibodies were procured from Cell Signaling (dilution 1:200). The VEGFR1 and SPRR1B antibodies were procured from Abcam. Anti-mouse and rabbit secondary antibodies were obtained from Jackson Immunoresearch (dilution 1:1000). All IHC and histological evaluation was done per pathologist's guidance.