Western Blot Analysis of Protein Expression

Cells and heart tissue were lysed in protein lysis (1% Deoxycholic acid, 10 mM Na₄P₂O₇, 1% TritonX-100, 10% Glycerol, 100 mM NaCl, 5 mM EDTA (pH = 8.0), 20 mM Tris-HCl (pH = 7.4), 0.1% SDS, 50 mM NaF, 1 mM Na₃VO₄, 1 mM PMSF, 10 mg/L aprotinin). Equal amounts of protein were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes. After blocking with 5% milk for 1 h, the membranes were incubated with primary antibodies at 4 °C overnight and incubated with secondary antibodies for 1 h at room temperature. After being treated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Abacm, ab97051, Cambridge, MA, USA), the protein bands were detected using a chemiluminescence reagent (Affinity, KF8003, San Francisco, CA, USA) and visualized on a GeneGnome chemiluminescent imaging system (Syngene, Iselin, NJ, USA) [9 (link)]. The relative density of each band was analyzed using ImageJ. β-Tubulin and GAPDH served as internal references in densitometric analysis. The primary antibodies used in this study are given in Table 2.