Total protein extraction kit

Total protein was extracted from hMSCs using the Total Protein Extraction kit (Sigma) according to the manufacturer’s protocols. The protein concentration was determined with a BCA Protein Assay kit (Thermo scientific, Rockford, Illinois, USA) according to the manufacturer’s instructions. Then 40 μg protein were separated by SDS-PAGE, and then were transferred onto polyvinylidene difluoride membrane. The membrane was incubated with mouse polyclonal antibodies specific for Wnt 11 (1:150; Santa Cruz, CA, USA) and β-catenin (1:1000; Santa Cruz) overnight at 4 °C. After washing with Tris-buffered saline containing 0.1 % Tween, the membranes were incubated with secondary antibody (1:5000; Santa Cruz) for 1 hour. Finally, the membrane was exposed, visualized using an enhanced chemiluminescent kit (Merck Millipore, Eschborn, Germany) and a chemiluminescence detection system (Bio-Rad, Hercules,California, USA). Protein bands were quantified using the Quantity One software (Bio-Rad).

Total proteins were extracted from the adipose tissue using the Total Protein Extraction Kit (Sigma, Cat# R0278). Protein concentrations were measured using the BCA Protein Assay Kit (Thermo scientific, Cat#23227). Twenty micrograms of proteins were resolved by 10% SDS-PAGE and transferred to PVDF membranes (Merck&millipore, Cat# SLGVV255F). Membranes were blocked with 5% bovine serum albumin (Merck&millipore, Cat#12659-500GM) in Tris-buffered saline with Tween 20 for 2 h followed by overnight incubation at 4°C with primary antibodies against UCP1 (Abcam, Cat#ab10983), PGC-1α (Santa Cruz, Cat#sc-13067), and PRDM16 (Abcam, Cat# ab106410). After three times washing with Tris buffered saline Tween20 (TBST), suitable HRP-labeled secondary antibody was incubated for 2 h at room temperature. Immunoblotting signals were visualized by ECL Kit (Thermo scientific). Bands were quantified by using the Image J software (NIH, Bethesda, MD, USA). Immunodetection of endogenous Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized to estimate that equal amounts of protein were present in samples.

Another cohort of WT and db/db mice treated with and without pGz was anesthetized (100 mg/kg of ketamine and 5 mg/kg of xylazine) and sacrificed by cervical dislocation. Gastrocnemius muscles were dissected, minced, and homogenized using a total protein extraction kit (Sigma Millipore, Saint Louis, MA, USA). Total protein concentrations were determined using the bicinchoninic acid (BCA) method (Thermo-Scientific, Waltham, MA, USA). Denatured, SDS-gel separated, and membrane-immobilized proteins were incubated overnight at 4 °C with primary antibodies: anti-eNOS, dilution 1:2500 (ab300072; Abcam, Waltham, MA, USA); anti-p-eNOS, dilution 1:2500 (ab230158, Abcam, MA, USA); anti-nNOS, dilution 1:2000 (ab76067, Abcam, MA, USA); anti-iNOS, dilution 1:2500 (ab283655, Abcam, MA, USA); anti-GAPDH, dilution of 1:5000 (SC47724; Santa Cruz, CA, USA); and secondary fluorescent antibodies (Abcam, MA, USA). The resolved bands were detected with a Storm 860 Imaging System (GE Bio-Sciences, Piscataway, NJ, USA). Protein levels were quantified using myImageAnalysis software V1.0 (Thermo-Fisher Scientific, Waltham, MA, USA) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH).