Horseradish peroxidase conjugated secondary antibody

MCF7 cell extracts were prepared in M-PER lysis buffer (Thermo Scientific) and adjusted to 5 mg / mL of total protein. Then, equal 500 μL samples were incubated with either a rabbit polyclonal anti-Hsp70 or Goat IgG and treated with either DMSO or JG-237 (10 µM). After rotating samples overnight at 4 °C and a 4 hr incubation with protein A/G-Sepharose Beads (Santa Cruz), the samples were centrifuged (1000 xg), washed with PBS pH 7.4, and eluted with SDS-loading dye. After separation, the gels were transferred and membranes were blocked in nonfat milk (5% milk in TBS, 0.1% Tween) for 1 hr, incubated with primary antibodies for Hsp70 and BAG3 overnight at 4 °C. Imaging was performed using a horseradish peroxidase-conjugated secondary antibody (Anaspec). Finally, membranes were developed using chemiluminescence (Thermo Scientific, Supersignal® West Pico).^{6 (link)}

The co-immunoprecipitation procedure was adopted from previously described methods (Thompson et al., 2012 (link)). Briefly, HeLa C3 cells were treated with bortezomib in a final concentration of 5 μM for 4 hours prior to lysis in mammalian protein extraction reagent (Thermo Scientific). Following protein quantification, 5 mg of lysate was incubated with 40 μL of goat anti-V5 conjugated to agarose beads (Bethyl Laboratories, S190-119) and 50 μM JG-48 or DMSO (5%) at 4 °C in the dark overnight. Lysate was incubated with normal goat IgG with DMSO (4%) as a negative control. Protein A/G agarose beads (20μL) (Santa Cruz, sc-2003) were added to samples for 4 hours. Beads were then washed 3 times with 100 μL of PBS (Gibco) at 1000×g for 1 minute. Proteins were eluted by boiling at 96°C in 40μL of 1× SDS loading dye. 10 μL of sample were separated on 10% Tris-trycine gels (Bio-rad) and transferred to nitrocellulose membrane. The membranes were blocked in nonfat milk (5% milk in TBS, 0.5% Tween) for 1 hour, incubated with primary antibodies for Tau (Santa Cruz, sc-5587) and Hsp72 (Enzo, ADI-SPA-811) overnight at 4 °C in the dark, washed, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Anaspec, 28177) for 1 hour. Finally, membranes were developed using chemiluminescence (Thermo Scientific, Supersignal® West Pico).