Rabbit anti pkm2 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.

All Western blot quantifications used 4 biological samples of 2-month-old mice with each sample consisting of both retinas from the same mouse. The analysis of each sample was performed in duplicate. Protein sample preparation and Western blot analysis were performed with the same reagents and techniques as previously described [14 (link)]. In brief, enucleated eyes were dissected in cold PBS buffer. Dissected retinas were immediately transferred into RIPA buffer (Thermo Scientific, cat# 89900) with protease and phosphatase inhibitors (1:100 dilution; cat#1861281) and homogenized by sonication. After 10 min centrifugation at 4 °C at 13,000 RPM, protein extracts were transferred into a fresh tube and protein concentration was quantified with the Bio-Rad Protein Assay (cat# 500-0113, 0114, 0115). To quantify PKM2 and p-S6 expression levels, 5 μg and 10 μg of total protein, respectively, were loaded. The following primary antibodies from Cell Signaling Technology were used: rabbit anti-PKM2 antibody (1:4000; Cat#4053), rabbit anti-pS6 (Ser240/244) (1:1000; Cat#5364), and for normalization, mouse anti-β-actin antibody (1:1000; Cat#3700). Protein detection was done using fluorescently labeled secondary (1:10,000) antibodies from Licor in combination with the Odyssey system. Quantification was performed with Image Studio software.