Rabbit anti v5 antibody

For immunoprecipitation experiments, mutant and wild-type14-3-3eta cDNAs were cloned into pcDNA/V5-His vector and transfected in Hela cells using Lipofectamine 2 000 (Invitrogen). Twenty-four hours after transfection, cells were lysed with 1% Triton lysis buffer (100 mM NaCl, 1% Triton, 0.02 M Tris, 0.005 EDTA) at 4 °C for 1 h. Mouse anti-Bad antibody (BD Transduction Laboratories, Franklin Lakes, NJ, USA) was previously coupled to protein-G Sepharose and used for immunoprecipitation. The presence of 14-3-3eta in the Bad complex was revealed by immunoblot with the rabbit anti-V5 antibody (Invitrogen).

After cell transfection, cells were washed with ice-cold PBS and lysed in lysis buffer A (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) + 1mM DTT (Dithiothreitol) + complete protease inhibitor cocktail (complete TM, Roche Diagnostics, Germany). For each reaction, A/G agarose beads (Protein A/G plus agarose, Santa Cruz Biotechnology) were incubated with rabbit anti V5 antibody (Invitrogen) in 20:1 ratio at 4 C° for 1 h with slow agitation (10 rpm) and washed once with lysis buffer before use. Cell lysates were centrifuged at 10000 rpm at 4 C° for 15 min, the supernatant was separated into a new tube and used as the input fraction. The input fraction was precleared first by incubating with A/G agarose beads at 4 C° for 1 h with slow agitation. Finally, the precleared input was incubated with anti V5-coated beads at 4 C° with slow agitation overnight. Input fractions were then centrifuged at 8000 rpm at 4 C° for 1 min and the resulting beads were washed 3–4 times with lysis buffer at the same centrifugal force. Proteins were dissolved in SDS sample buffer and boiled for 5 minutes. SDS-PAGE and western blotting were performed as mentioned before using mouse anti-myc and rabbit anti V5 antibodies to detect HSPB5 mutants and HSPB4 respectively.