Sc 17808

Transfected cells were harvested with RIPA buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl (pH 8.0), 0.1% SDS, 0.5% sodium deoxycholate, and 1X protease inhibitor cocktail set III (Calbiochem)) 2 days after transfection. Immunoprecipitation was performed with mouse anti-Myc (9E10, Santa Cruz) antibody or anti-Flag-M2 (F3165, SIGMA) antibody. The antibodies were removed by incubation with protein G Sepharose (Zymed/Invitrogen), and the wash step was repeated 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). To examine the interaction of Flag-C16orf74 with Myc-PPP3CA, we analyzed the immune complexes by western blotting with rabbit anti-Flag and anti-Myc antibodies. To examine the interaction of endogenous C16orf74 with endogenous PPP3CA, Capan-1 cells were harvested in RIPA buffer, and immunoprecipitation was performed with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody or rabbit anti-C16orf74 (immunized with full-length recombinant C16orf74 protein) antibodies. Then, we collected the antibodies with protein G Sepharose (Zymed/Invitrogen) and repeated the wash steps 5 times. Proteins were extracted with SDS sample buffer and separated by 10-20% gradient SDS-page (Bio-Rad). We analyzed the immune complexes by Western blotting with mouse anti-PPP3CA (sc-17808, Santa Cruz) antibody, or rabbit anti-C16orf74 antibodies.

The cultured cells were washed twice with PBS and harvested in lysis buffer (150 mM NaCl, 1% Triton X-100, 50 mM Tris-HCl pH 7.4, 1 mM DTT, and 1X complete Protease Inhibitor Cocktail (Boehringer)). After the cells were homogenized and centrifuged at 10,000xg for 30 min, the protein concentration of the supernatant was standardized by the Bradford assay (Bio-Rad). Proteins were separated by 12% SDS-PAGE and immunoblotted with antibody. The antibodies used in the western blot analysis were rat anti-HA, mouse anti-HA (sc-7392, Santa Cruz), anti-Flag-M2, rabbit anti-Flag (F7425, SIGMA), mouse anti-beta-actin (A5441, SIGMA), mouse anti-PPP3CA (sc-17808, Santa Cruz), and rabbit anti- C16orf74 antibodies.