Westernbright sirius ecl kit

For immunoprecipitation experiments, total homogenates from adipose tissue and cultured cells were treated with RIPA lysis buffer (P0013B, Beyotime Biotechnology, China), vortexed for the 30s, and centrifuged for 15 min at 12000 r/min. The tissue or cell extracts was subjected to immunoprecipitation with HIF1α primary antibody at 4°C overnight. The antibody-bound proteins were precipitated with 20 μL protein A/G PLUS-Agarose (Santa Cruz Biotechnology, sc-2003) and rotated for 1 h, then incubated overnight at 4°C. The beads were then gently centrifuged at 1000 r/min for 5 minutes at 4°C. After four RIPA buffer washes, the immunoprecipitates were diluted with 40 μL of 1 × SDS loading buffer (CW0027, Cowin Biotech, China) and boiled at 100°C for 2-3 min to separate complexes from the protein A/G PLUS-Agarose. The samples were then subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, USA). After blocking with QuickBlock™ Western (P0252, Beyotime Biotechnology, China), the membranes were incubated with an anti-acetylated-lysine antibody (Cell Signaling Technology, #9441) 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).

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.