Quantifying Protein Expression in Cardiac Tissue

The BCA kit (Cwbio, Jiangsu, China) was utilized to quantify the protein isolated from cardiac tissues, which were further separated with the 12% SDS-PAGE. The separated protein was then transferred from the gel to the PVDF membrane, and mixed with 5% skim milk. Then, the membrane was introduced with the primary antibody against p-JAK-2 (1:1000, Santa Cruz Biotechnology, USA), JAK-2 (1:2000, Santa Cruz Biotechnology, USA), p-STAT-3 (1:1000, Santa Cruz Biotechnology, USA), STAT-3 (1:2000, Santa Cruz Biotechnology, USA), and β-actin (1:8000, Santa Cruz Biotechnology, USA). The secondary antibody (1:2000, SolelyBio, Beijing, China) was subsequently added to be incubated for 90 min. Finally, the ECL reagent was added to expose the bands, which were further quantified with the Image J software [16 (link)].