Cdk6 antibody

Cells were lysed using mammalian protein extraction reagent RIPA (Beyotime china) supplemented with protease inhibitors cocktail (Roche, Switzerland) and PMSF (Roche, Switzerland). Protein concentration was measured with the Bio-Rad protein assay kit. 50 μg protein extractions were separated by 12% SDSpolyacrylamide gel electrophoresis (SDS-PAGE), then transferred to 0.22 μm nitrocellulose membranes (Sigma-Aldrich. USA) and incubated with specific antibodies. ECL chromogenic substrate was used to visualize the bands and the intensity of the bands was quantified by densitometry (Quantity One software; Bio-Rad). GAPDH was used as control. GAPDH antibody was purchased from sigma-Aldrich (USA), and CDK6 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Immunohistochemistry (IHC) was performed using the SP kit (ZSGB-BIO, Beijing, China), in accordance with the manufacturer's instructions. Tissue sections were incubated with a CDK6 antibody (Santa Cruz; 1:200) and antibody binding was developed using a DAB chromogenic kit (Beyotime, Nanjing, China). For analysis, the sections were randomly numbered, double-blinded and analyzed by light microscopy (E100; Nikon). Analysis was performed independently by two pathologists; their consensus was considered as the final score. The extent of positive staining was scored by calculating the proportion (%) of positively stained cells: <5%, 0 points; 5-10%, 1 point; 10-50%, 2 points and >50%, 3 points. The intensity of staining was scored as follows: no staining, 0 points; light yellow, 1 point; moderate yellow, 2 points and strong yellow, 3 points. The final IHC score was determined by multiplying the extent of the staining by the intensity; scores of 0-3 and 4-9 were considered negative and positive expression, respectively (Lv et al. 2018) (link).