Heat shock protein 27

The cells were lysed in 10 mM Tris-HCl (pH 7.4) containing 3 mM MgCl₂, 10 mM NaCl, 0.1% SDS, 0.1% Triton X-100, 0.5 mM EDTA and protease inhibitors, separated on SDS-PAGE and transferred to nitrocellulose sheets at 400 mA for 2 h at 4 °C. Western blot analysis was performed using antibodies against TRPM7 (Bethyl, Montgomery, AL, USA), MagT1 (Abcam, Cambridge, UK), SLC41A1, Mrs2, cyclophilin D (CypD), thioredoxin-interacting protein (TXNIP), paraoxonase 2 (PON2) and superoxide dismutase 2 (SOD2) (Thermo Fisher Scientific, Waltham, MA, USA), heat shock protein (Hsp) 27 (Cell Signaling Technology, Danvers, MA, USA), Hsp70 and actin (Santa Cruz Biotechnology, Dallas, TX, USA). After extensive washing, secondary antibodies labeled with horseradish peroxidase (GE Healthcare, Waukesha, WI, USA) were used. Immunoreactive proteins were detected by the SuperSignal Chemiluminescence Kit (Thermo Fisher Scientific, Waltham, MA, USA). All the experiments were performed three times, and a representative blot is shown. Densitometry of the bands was performed with the software ImageLab (Bio-Rad, Hercules, CA, USA) on three blots ± (SD).