Goat anti mouse immunoglobulin g igg and goat anti rabbit igg secondary antibodies

After different treatments, the cells were washed twice with PBS and lysed in buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mg/mL leupeptin, 1 mM phenylmethyl-sulfonylfluoride). Protein concentration was measured by the Bradford method. Equal amounts of protein (50 μg) were loaded onto 10% SDS-PAGE gels and blotted onto a polyvinylidene fluoride membrane. To block the membrane, 5% fat-free milk was added, followed by incubation with primary antibody for 1 h and secondary antibody for 1 h at room temperature. Western blot detection was carried out using a Li-Cor Odyssey image reader (Li-Cor, Lincoln, NE, USA). Goat anti-mouse immunoglobulin G (IgG) and goat anti-rabbit IgG secondary antibodies were obtained from Li-Cor. The following antibodies were used in this study: PKM1 (Cell Signaling Technology, #7067at 1:1000 dilution), PKM2 (Cell Signaling Technology, #4053at 1:1000 dilution), and β-actin (Santa Cruz, Cat #sc-1616at 1:5000 dilution) as an internal control.