Mouse anti cyclin d1

Protein concentration was determined by Bradford method (Thermo Fisher Scientific). For determination of expression levels of protein in lysed cells, the protein was separated by SDS‐PAGE using 10% acrylamide gel. Then, the separated proteins were transferred onto nitrocellulose membrane and blocked with 5% (w/v) skim milk in 1× TBS containing 0.3% (v/v) Tween (TBS‐T) at room temperature for 1 hour. Membranes were washed twice with TBS‐T and incubated with the primary antibody, mouse anti‐cyclin D1 (Merck Millipore, Danvers, MA, USA) and rabbit anti‐survivin (Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. After washing five times with TBS‐T, membranes were incubated with the HRP‐conjugated anti‐rabbit or mouse IgG depending on primary antibody (GE Healthcare Bio‐sciences, Buckinghamshire, NA, UK) at room temperature for 2 hours and washed with TBS‐T five times. Immunoreactive material was then visualized with an enhanced chemiluminescence detection system (GE Healthcare Bio‐sciences). To confirm equal protein loading, each membrane was stripped and incubated with mouse anti‐β‐actin (Sigma‐Aldrich).