4.2. Co-Immunoprecipitation and Western Blotting

To detect phosphorylated HSF1, HEK293 cells were transiently transfected with Flag-HSF1-FL (full-length) and Flag-HSF1-CT (C-terminal domain), respectively, and then transfected HEK293 cells were further incubated in the absence or presence of {"type":"entrez-protein","attrs":{"text":"P22077","term_id":"134707","term_text":"P22077"}}P22077. Total cell lysates were obtained by using a lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl (pH 7.9), 0.1 mM ethylenediaminetetraacetic acid (EDTA), and a protease inhibitor cocktail. Cell lysates (2 mg/mL) were incubated with 1 μg of antibody-recognizing phosphorylated-serine for 16 h at 4°C, and then the immune complexes were gently washed with wash buffer containing 0.5% NP-40, 200 mM of NaCl, 50 mM of Tris-HCl (pH 7.9), 0.1 mM of EDTA, and protease inhibitor cocktail. The immunoprecipitated protein complex was dissolved with SDS (sodium dodecyl sulfate) sample buffer (50 mM of Tris-HCl pH6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, and 12.5 mM of EDTA, 0.02% bromophenol blue). For Western blotting, total protein was extracted from the cells with NP-40 lysis buffer containing 1% NP-40, 2.5M of NaCl, 1M of Tris-HCl, 500 Mm of EDTA, 1 M of NaF, and protease inhibitor cocktail. A Bradford protein assay was conducted to determine total protein concentration. Total proteins were resolved by SDS (sodium dodecyl sulfate)-PAGE (polyacrylamide gel electrophoresis), then transferred onto PVDF membranes (Millipore, Burlington, MA, USA) and blocked with 5% skimmed milk (w/v) in TBST buffer incubated with the indicated primary antibodies (1:1000) overnight at 4 °C. The membranes were further incubated with the corresponding HRP-conjugated secondary antibodies (1:10,000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at room temperature. Antibodies against phosphorylated serine (Cat# 612546), β-actin (Cat# sc-47778), Flag-tag (Cat# F3165), p-eIF2α (Cat# 9721), HSF1 (Cat# 12972), and ubiquitin (Cat# sc-8017) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), Sigma-Aldrich (St. Louis, MO, USA), and Cell Signaling Technology (Danvers, MA, USA). Finally, protein expression was visualized using an ECL Prime kit (GE Healthcare, Milwaukee, WI, USA).