Mouse monoclonal anti vcp antibody

Recombinant mouse PKM2 (100 pmol) and recombinant human VCP (10 pmol) were coincubated for 1 h at 25 °C. Fifty µl of Dynabeads Protein G (Thermo Fisher Scientific) were treated with 1% bovine serum albumin for blocking in 0.01% Tween phosphate buffered saline for 30 min at 4 °C with rotation. Then, the protein G was incubated with a rabbit anti-PKM2 antibody (2 µL, Cell Signaling Technology, Danvers, MA, US) or normal mouse IgG (2 µL, Santa Cruz Biotechnology, Dallas, TX, US) for 30 min at 4° with rotation. Protein G and antibodies were cross linked by 50 mM dimethyl pimelimidate dihydrochloride (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) for 1 h at 25 °C with rotation. After washing protein G, incubated recombinant proteins were added, and the mixture was incubated for 2 h at 25 °C with rotation. For the elution of immunoprecipitants, the samples were mixed with LDS sample buffer and 0.1 M glycine–HCl (pH 2.8) for 5 min at 95° and then were loaded onto 10% polyacrylamide gels and electrophoresed. After electrophoresis, PKM2 or VCP in the gel were detected by Western blotting using a rabbit monoclonal anti-PKM2 antibody (1:1000, Cell Signaling) or a mouse monoclonal anti-VCP antibody (1:2000, Abcam) as first antibodies. Secondary antibodies against rabbit IgG (1:2000, Cell Signaling) and mouse IgG (1:2000, Abcam) were used.

Western blotting was performed using samples after proteolysis in the DARTS analysis^{11 (link)}. Lysates of the plasma membrane fraction of cultured neurons were prepared with the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) after four days of in vitro culturing following the manufacturer’s protocol. Samples from the DARTS reaction were loaded onto a 10% SDS-PAGE. After electrophoresis, proteins in the gel were transferred to a nitrocellulose membrane (Bio-Rad, Berkeley, CA, United States) and blocked with 0.1% T-TBS containing 5% skim milk (Wako) at 24 °C. Subsequently, the membrane was incubated with a mouse monoclonal anti-VCP antibody (1:5000 for Fig. 1, Abcam) after blocking. The reaction was done in Can Get Signal solution 1 (Toyobo, Osaka, Japan) for 18 h at 4 °C. After washing, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody against mouse IgG (1:2000, Abcam) in Can Get Signal solution 2 (Toyobo) for 2 h at 24 °C room temperature. Chemiluminescence of protein bands were detected by ECL Prime Western Blotting Detection Reagent (GE Healthcare) using an ImageQuant LAS 4000 system (GE Healthcare).