Immunoblotting and qPCR Analysis of eWAT and SVF

Immunoblotting analysis and qPCR were performed according to previous articles (29 (link)). In brief, Protein-extracts of snap-frozen eWAT and whole-cell lysates of SVF were prepared using standard procedures. Protein concentrations in the supernatants were measured using Bicinchoninic acid (BCA) assay (ASPEN, USA). Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. After blocking with QuickBlock™ Western (P0252, Beyotime Biotechnology, China), the membranes were incubated with the primary antibodies overnight at 4°C, washed in PBST three times, and incubated with a secondary goat anti-rabbit polyclonal antibody (SA00001-2, Proteintech Group) at room temperature for 1h. Finally, the signals were tested by WesternBright™ Sirius ECL kit (K-12043-D20, Advansta, USA). Protein expression levels were normalized to β-actin.
Total mRNA was extracted from eWAT or SVF cells with GeneJET RNA Purification Kit (K0731, Thermo Fisher Scientific, USA). Reverse transcribed into cDNA using RevertAid First strand cDNA Synthesis kit (K1622, Thermo Fisher Scientific, USA). The StepOne Real-Time PCR (Life tech, Alameda, CA) was used for real-time qPCR analysis. The primers used are described in Table S1. β-actin was used as an internal control. The relative expression quantity 2^-ΔΔCt value was calculated to compare the differences among groups.