Protein Extraction and Western Blot Analysis

To extract proteins from primary, K4D, and K4DT + T cells, we lysed cells in a buffer containing 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 1% Triton X-100, 2.5 mg/ml sodium deoxycholate (Wako, Osaka, Japan; cat. no. 194-08311), and a protease inhibitor cocktail (1/200 dilution, Nacalai Tesque; cat. no. 25955-11). The protein expression levels of CDK4 and Cyclin D were detected by Western blotting using an anti-CDK4 antibody (1/2,500 dilution, MBL, Nagoya, Japan; cat. no. 25955-11), an anti-cyclin D antibody (1/5,000 dilution, MB; cat. no. 553), and an anti-alpha-tubulin antibody (1/1,000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA; cat. no. sc-32,293). Anti-mouse IgG (1/2,000 dilution, GE Healthcare, Buckinghamshire, UK; cat. no. NA931) or anti-rabbit IgG (1/2,000 dilution; GE Healthcare; cat. no. NA934-1ML) was used as secondary antibodies. The detailed Western blot procedure has been reported previously (Fukuda et al., 2005 (link), 2008 (link)). The intensity of signals was measured by the Image J software.