Anti glucose regulated protein 78

After treating bladder cancer cells under the indicated conditions for 48 hours, whole cell lysates were obtained using radioimmunoprecipitation assay buffer. Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. After the membranes were blocked with 5% skimmed milk, they were incubated with the primary antibodies: anti-AMPK and anti-PPARγ from Proteintech (Rosemont, IL, USA); anti-phosphorylated AMPK (p-AMPK), anti-phosphorylated histone H2AX (p-H2AX), and anti-endoplasmic reticulum resident protein (ERp) 44 from Cell Signaling Technology (Danvers, MA, USA); anti-glucose-regulated protein (GRP) 78, anti-cyclin D1, anti-cyclin E, anti-cyclin-dependent kinase (CDK) 2, anti-CDK4, anti-HDAC1, anti-HDAC3, and anti-HDAC6 from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-acetylated histone from Abcam (Cambridge, UK); and anti-actin from Millipore (Billerica, MA, USA). Then the protein was detected by reaction with recommended secondary antibody (horseradish-tagged goat anti-rabbit or goat antimouse antibody (GE Healthcare UK, Amersham, UK)) and staining with chemiluminescence solution (Clarity Western ECL Substrate, Bio-Rad, Hercules, CA, USA) and imaged with ChemiDoc Touch Imaging System (Bio-Rad).