Ls b243

Mouse monoclonal antibodies for CITED2 (LS‐B243) and Ki‐67 (MIB1) were purchased from LSBio (Seattle, WA, USA) and Dako (Carpinteria, CA, USA), respectively. The antigen–antibody complex was visualized with 3,3′‐diaminobenzidine solution and counterstained with hematoxylin. Immunohistochemistry for ER (CONFIRM anti‐ER [SP1]) and progesterone receptor (PR) (CONFIRM anti‐PR [1E2]; Roche Diagnostics Japan, Tokyo, Japan) was performed with Ventana Benchmark XT (Roche Diagnostics Japan), and that for HER2 was performed by HercepTest (Dako).

Total protein was extracted using M‐PER (Thermo Fisher Scientific Pierce Biotechnology, Rockford, IL, USA). The lysate proteins (15 μg) were subjected to SDS–PAGE (10% acrylamide gel) and transferred onto Hybond PVDF membranes (GE Healthcare, Buckinghamshire, UK). The primary anti‐CITED2 antibody used was the same as that in the immunohistochemistry (LS‐B243, LSBio), and anti‐p53 antibody (DO‐1) was purchase from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti‐β‐actin antibody (A3854, Sigma‐Aldrich) was also used as an internal control. In this study the p53 protein level was calculated as the ratio of the β‐actin protein level.