Goat anti mouse igg hrp conjugated sc 2005

Mouse polyclonal anti-TROAP antibody (SAB1406913) was purchased from Sigma (Sigma-Aldrich, St. Louis, MO, USA). Mouse anti-human GAPDH antibody (sc-32233), HRP-conjugated goat anti-rabbit IgG (sc-2004), and HRP-conjugated goat anti-mouse IgG (sc-2005) were purchased from Santa Cruz Biotechnology, Inc. TRIzol reagent and an ECL Substrate Reagent Kit were purchased from Invitrogen (Grand Island, NY, USA). Western blotting (WB) equipment and materials were purchased from Sigma-Aldrich (St. Louis, MO, USA).

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.

Western blot analyses were performed as described by Bastida-Ruiz et al. [24 (link)]. Briefly, whole-cell extracts were fractionated by SDS-Page 10% and transferred to nitrocellulose membrane for immunoblot analysis. The membranes were first incubated with the primary antibodies rabbit anti-GRP78 (GL-19, 1∶5000 dilution from Sigma) and mouse anti-GAPDH antibodies (1∶60,000 dilution from Millipore), followed by incubation with the secondary antibodies goat anti-rabbit IgG horseradish peroxidase (HRP)-conjugated (170-6515, 1:3000 dilution, from Bio Rad) and goat anti-mouse IgG HRP-conjugated (sc-2005, 1:3000 dilution, Santa Cruz Biotechnologies).