Anti tra2β

Whole-cell lysates were prepared in a RIPA buffer (10 mm Tris-HCl, pH 7.4; 1% Nonidet P-40; 1 mm EDTA; 0.1% sodium dodecyl sulfate; 150 mm NaCl) containing a protease and phosphatase inhibitor cocktail (Roche Diagnostics Japan, Tokyo, Japan). Mouse monoclonal anti-α-tubulin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tra2β (1:1000; Abcam, Cambridge, UK), anti-caspase-3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-cleaved caspase-3 (1:1000; Cell Signaling Technology), anti-PARP (1:1000; Cell Signaling Technology), anti-cleaved PARP (1:1000; Cell Signaling Technology), anti-p21 (1:1000; Santa Cruz Biotechnology), anti-p53 (1:1000; Santa Cruz Biotechnology), anti-β-actin (1:1000; Abcam) or anti-Sp1 (1:1000, Santa Cruz Biotechnology) antibody was used.

3x10⁵ HEK293T cells were transfected with plasmids using a calcium phosphate protocol [31 ]. Forty-eight hours posttransfection, cells were washed and harvested in PBS. The cells were pelleted by centrifugation at 5000 rpm for 5 minutes and the pellet was resuspended in 30 μl of SDS-PAGE sample buffer (0.125M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.2% bromophenol blue). The samples were boiled for 4 minutes and fractionated on 12.5% SDS-PAGE gels. Proteins were transferred onto PVDF membranes, the resulting blots washed with 0.05% Tween in PBS and blocked with 5% milk in PBS for 30–60 minutes. Proteins were detected using anti-HIV-1 p24^CA hybridoma supernatants [32 (link)], anti-HIV-1 Env hydriboma antibody [33 (link)], anti-tubulin (Sigma), anti-Tra2β (Abcam, ab31353), or anti-myc (9E10) antibodies followed by HRP-conjugated anti-mouse antibody (Jackson Laboratories) and the signal developed using Western Lightning Plus (Perkin Elmer Life Sciences). To assess the relative level of transfected and endogenous proteins, blots were also probed with rabbit anti-Tra2β antibody generously provided by Stephan Stamm.