Bca protein determination 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).

Protein refolding activity was examined with luciferase inactivation assay with minor modifications [72 (link)]. In brief, 100 nM luciferase (Sigma-Aldrich Cat#61970-00-1) was heated at 42 °C alone or in the presence of the fly muscle lysates in luciferase refolding buffer (LRB: 25 mM HEPES-KOH at pH 7.4, 150 mM potassium acetate,10 mM magnesium acetate and 10 mM DTT). Luminescence was measured on a microplate reader (Synergy HTX, BioTek, Winooski, Vermont, USA) at different time points (5, 10, 15 min). Luminescence values were then normalized with protein concentrations using BSA as a standard by bicinchoninic acid (BCA) protein determination kit (YEASEN Cat#20201ES76) according to the manufacturer’s instructions.

U-GLAD system was utilized for large-scale compound screening in flies [19 ]. In brief, a compound library containing 1508 FDA approved drugs and 345 natural products (DiscoveryProbe^™ FDA-approved Drug Library, ApexBio Cat# L1001) were individually mixed in Gum Arabic and dissolved in chemically defined liquid food to form micelles (final concentration: 5 mg/ml). These micelles are then delivered to CRE-LUC flies in vials by a U-shape glass capillary. Chemically defined food recipe was based on previous study [37 (link)]. CRE-Luciferase activity was measured using the Steady-Glo Luciferase Assay Kit (Promega). Protein concentrations were determined with BSA as a standard using a bicinchoninic acid (BCA) protein determination kit (YEASEN Cat#20201ES76) according to the manufacturer’s instructions.