Anti omi htra2

Myocardium tissues were harvested for Western Blot following standard protocol. Nucleoprotein was extracted according to the Nuclear and Cytoplasmic Protein Extraction Kit (P0027, Beyotime Institute of Biotechnology, Suzhou, China). 50 μg/lane protein or 20 μg/lane nucleoprotein Proteins were run on 10% SDS-PAGE, and then electrotransferred onto polyvinylidene difluoride membranes. The membranes were incubated with the primary antibodies anti-HSF1(#4356; 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-Omi/HtrA2(#2176; 1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-GAPDH(#2118; 1:1000; Cell Signaling Technology, Danvers, MA, USA), or anti-Histone H3 (#4499; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4°C. After incubation with the corresponding secondary antibodies, the membrane was developed with a chemiluminescent substrate (Bio-Rad), and protein bands were measured using the ImageJ software.

Protein lysates from tissues were prepared as previously described [13 (link)], and protein concentrations of lysates were determined using a BCA Protein Assay Kit (#23225; Thermo Scientific Pierce, Carlsbad, CA, USA). Fifty µg/lane of each sample were run on 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gels, depending of the target proteins, and then were electrotransferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies anti-Omi/HtrA2 (#2176; 1:1000; Cell Signaling Technology, Danvers, MA, USA) or rabbit anti-glyceraldehyde-3-phosphate dehydrogenase/GAPDH (#2118; 1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C, followed by incubation with the matched secondary antibodies for 1h at room temperature. The density of the target protein bands was measured using ImageJ software, and GAPDH was used as a protein loading control.