Proper horseradish peroxidase conjugated secondary antibodies

Western blot was performed following previously published method [29 (link)] to analyze the changes of specified proteins. Briefly, primary skeletal muscle fibroblasts were lyzed with RIPA buffer (Beyotime Bio, Shanghai, China) supplemented with proteinease inhibitor and phosphatase cocktail (Sigma, St. Louis, MO) and total proteins (40 μg) were resolved on 8% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked in 5% nonfat milk in TBST (50 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) for 45 min, incubated with primary antibodies at 4 °C overnight, washed and incubated with proper horseradish peroxidase conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) at room temperature for 60 min before visualized with enhanced chemiluminescence (ECL) reagents (Pierce, Rockford, IL). The primary antibodies used were Col I antibody, a-SMA antibody, CTGF antibody, TGF-β1 antibody, Fibronectin antibody, p-AKT antibody, AKT antibody, p-ERK antibody, PKC antibody, p-PKC antibody and β-Actin Antibody (sc-47,778) were purchased from Santa Cruz Biotech (Shanghai, China).

The HUVECs were lyzed in 100 μL of RIPA buffer (50 mM Tris-cl pH 7.4, 150 mM NaCl, 1% NP40, and 0.25% Na-deoxycholate) containing 1x complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and 1x phosphatase inhibitor cocktail (Sigma, St. Louis, MO) on ice for 30 min. After centrifugation at 10000 ×g at 4°C for 15 min, the supernatant was collected and protein concentration was measured with Pierce Coomassie Plus (Bradford) Assay Kit (23236, ThermoFisher). 30 μg total protein was resolved in a 8% sodium dodecyl sulfate polyacrylamide gel and transferred onto a PVDF membrane, which was blocked in 5% nonfat milk in PBST (0.1% Tween 20 in PBS) at room temperature for 30 min followed by being incubated with specified first antibodies at 4°C overnight. Next day, the membranes were washed 3 times at 5 min each time with PBST and then incubated with proper horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) for 60 min at room temperature. The specific bands were detected with Pierce ECL Plus Substrate (ThermoFisher). The primary antibodies used were p-NF-κB p65 (ab86299), NF-κB p65 (ab16502), ICAM-1 (ab124759), and LOX-1 (ab60178) from Abcam (Cambridge, MA), p-p38MAPK (4511), and p38MAPK (8690) from Cell Signaling (Shanghai, China) and β-actin (TA-09) from ZSGB-Bio (Beijing, China).