Anti γ tubulin antibody c 20

Whole-cell lysis, protein extraction and Western Blot analyses of cultured K562^shScr, K562^shFHC, K562^cntr, and K562^siFHC cells were performed, as previously reported [25 (link),26 (link)]. For FHC protein quantification, the incubation of the anti-rabbit polyclonal primary anti-FHC (H-53) (1:200; sc-25617, Santa Cruz Biotechnology, Dallas, TX, USA) followed by the incubation with the HRP-conjugated secondary antibody (1:3000 Cell Signaling) was carried out. The goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology) was used as loading control. The immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology) and the bands intensity was quantified by using ImageJ (National Institutes of Health, Bethesda, MD, USA).

Total cell lysates were prepared using RIPA buffer^{24 (link),26 (link)}. Each protein sample (40–50 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes^{24 (link),26 (link)}. Membranes were incubated with primary antibodies at 4 °C overnight. Antibodies against FHC (B-12) (1:200, sc-376594), p53 (A-1) (1:500, sc-393031), BAX (B-9) (1:500, sc-7480), and Bcl-2 (100) (1:500, sc-509) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, Texas); antibodies against Caspase-9 (1:500, #9502), Caspase-8 (1C12) (1:500, #9746), and Fas (C18C12) (1:500, #4233) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). Membranes were then incubated with secondary antibodies, HRP-conjugated goat anti-mouse IgG (1:2000, sc-516102; Santa Cruz Biotechnology, Dallas, Texas) and HRP-conjugated mouse anti-rabbit IgG (1:2000, sc-2357; Santa Cruz Biotechnology, Dallas, Texas), and immunoreactive bands were visualized with the ECL western blotting detection system (Santa Cruz Biotechnology, Dallas, Texas). To ensure equal loading of proteins a goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396; Santa Cruz Biotechnology) was used. The protein band intensity on western blots was quantified and normalized to that of γ-Tubulin by using ImageJ software (http://rsb.info.nih.gov/ij/).

Total cell lysates were prepared using RIPA buffer^{38 (link),39 (link)}. Each protein sample (40–60 μg) was separated by 10–15% SDS–PAGE and then transferred to nitrocellulose membranes. Membranes were incubated with primary antibodies at 4 °C overnight. Primary antibodies against c-Myc (C33, sc-42), Cyclin D2 (B-6, sc-376676), Vimentin (V9, sc-6260), SNAI1 (E-10, sc-393172), SLUG (A-7, sc-166476) and Bcl-2 (sc-509) were purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, Texas (Santa Cruz Biotechnology, Dallas, Texas) while antibodies against Fas (4233 S) and Caspase 3 (9662 S) were purchased from Cell Signalling Technology, Leiden, Netherlands). Membranes were then washed and incubated, for 2 h, with secondary antibodies HRP-conjugated goat anti-mouse IgG (sc-2005) and HRP-conjugated goat anti-rabbit IgG (sc-2357) (Santa Cruz Biotechnology, Dallas, Texas), and immunoreactive bands were visualized with the ECL Western blotting detection system (Santa Cruz Biotechnology, Dallas, Texas). To ensure equal loading of proteins was used a goat polyclonal anti-γ-Tubulin antibody (C-20) (1:3000; sc-7396, Santa Cruz Biotechnology). Experiments were performed three times and representative images are reported.