Electrophoretic Mobility Shift Assay

Binding of HSF-1 to its genomic target sequence was measured by Electrophoretic Mobility Shift Assay (EMSA) using a previously described protocol (Chiang et al. 2012). Briefly, 1 μg of nuclear extract was incubated for 20 min at room temperature in EMSA binding buffer (10 mM Tris-Cl at pH 7.5, 50 mM KCl, 1 mM DTT) supplemented with 50 ng/μL Poly (dI⋅dC) and 1 nM biotin-TEG-labeled oligonucleotide containing an HSE sequence. The biotin-TEG-labeled oligonucleotide was synthesized by annealing two complementary sequences (listed in Table S3) corresponding to an HSE from the promoter region of hsp-16.1 gene. The DNA-bound nuclear extracts were resolved using native 3.5% polyacrylamide gel electrophoresis and were transferred to Immobilon-Ny+ charged nylon membrane (Millipore Sigma) at 400 mA for 1 hr on a Trans-blot SD semi-dry transfer cell (Bio-Rad). The HSF-1-HSE DNA complexes were visualized by autoradiography using LightShift Chemiluminescent EMSA kit (Thermo Fisher).