Western Blot Analysis of Phosphorylated STAT3 in Heart Tissue

Whole ventricle tissues or infarcted areas of heart tissues were homogenized in lysis buffer containing 25 mM HEPES (pH 7.5), 1% Triton X 100, 150 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). The tissue extracts were resolved by the Nu-PAGE system (Thermofisher Scientific, Waltham, MA), and Western blot analysis performed as described previously [17 (link), 21 (link)]. Antibodies against tyrosine-phosphorylated STAT3 (p-STAT3; #9145, 500 ng/mL) and STAT3 (#12640, rabbit polyclonal, 23 ng/mL) were purchased from Cell Signaling Technology (Danvers, MA). Antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; MAB374, 6C5, 200 ng/mL) was purchased from Merck Millipore (Darmstadt, Germany). Antibody against IL-22R1 (ab5984, 1 μg/mL) was purchased from Abcam (Cambridge, United Kingdom). Antibody against IL-10RB (15102-1-AP, 450ng/mL) was purchased from Proteintech (Chicago, IL).