Anti bid

Cells were infected with various viruses and cells were lysed at 24 hr post-infection and protein complexes were isolated using anti-Flag Ab coated agarose beads. The proteins bound to the beads were washed 4 times and suspended in 40 μl sample buffer. The lysates and immunoprecipitated samples were separated by 4-12 % gradient gels, transferred to nitrocellulose membrane and incubated at 4°C overnight with the following antibodies; anti-Flag (Sigma), anti-BIK, anti-prohibitin 1 and 2 and anti-actin (Santa Cruz), anti-DNA-PKcs and anti-ATP synthase (Protein Tech), anti-BAK and anti-BAX (Upstate), anti-BIM, anti-BID and anti-PARP (BD Pharmingen). The blots were washed and incubated with the corresponding secondary antibodies conjugated with HRP (Santa Cruz) for 30 min. The blots were then washed and the protein bands were visualized with western blotting detection system (Roche) according to the manufacturer's specifications. The quantitation of cleaved PARP was carried out using BIO-RAD CHEMI DOC XRS system by measuring the relative intensities of the cleaved products.

Twenty-four and forty-eight hours after treatment of PC-3 and DU145 cells with 0 or 5 μM of DMAPT, or 0 or 4 μM of DMAPT, respectively, cells were analyzed for changes in protein expression by our standard Western analysis [26 (link),28 (link),46 (link)]. The primary antibodies utilized were anti-p53 (Ab-6, Calbiochem and clone BP53–12 Upstate), anti-p73 (rabbit polyclonal, Chemicon), anti-p21^{waf−1/Cip−1} (clone 6B6, BD Pharmingen and mixed monoclonal, Upstate), anti-Bax (Clone YTH-2D2, Trevigen), anti-Bcl-2 (clone 124, Dako), anti-Bid (BD Pharmingen), and anti-α-tubulin (cloneB-5–1–2, Sigma). The membranes were incubated with appropriate secondary antibodies conjugated with peroxidase (Pierce, Rockford, IL).