Ecl detection kit

We collected wound tissues for total protein extraction and analyzed STAT3 and p-STAT3 levels after 3 days of cutaneous-wound healing in vivo. Traumatized cutaneous tissues from mice were scraped and resuspended in 0.5 ml RIPA; tissue lysates were placed on ice for 1 h. After centrifugation at 10,000g for 20 min, we determined total protein concentration using a Bicinchoninic Acid (BCA) Protein Determination Kit (Yeasen). We electrophoresed 20-ct samples via 8% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; Yeasen) and transferred them onto the PVDF membrane. Samples were blocked with blocking buffer at RT for 2 h and then probed with STAT3 (dilution, 1:500; Invitrogen), p-STAT3 (dilution, 1:1000; Invitrogen), and β-actin (dilution, 1:10000; AB clonal Technology, Woburn, MA, United States) at 4°C overnight. Secondary aBs used for detection included horseradish peroxidase (HRP)–conjugated anti-rabbit immunoglobulin G (IgG; dilution, 1:10000; ABclonal Technology). We detected target protein expression using an Enhanced Chemiluminescence (ECL) Detection Kit (Boster).

Total protein extracts were prepared, and the protein concentration was measured as described previously 18 (link). Generally, 15 μg protein was separated by 10% SDS-PAGE and transferred onto PVDF membrane, then blocked with 5% fat-free milk and incubated with primary antibodies and HRP- conjugated secondary antibodies. The protein levels were detected by using the Enhanced Chemiluminescent (ECL) Detection Kit (Boster, Cat# EK1001).