Phospho hsl

Western blot analysis was performed as previously described by Pacifici et al. [21 (link)]. Briefly, cells were lysed at 4°C in HNTG lysis buffer (1% TritonX-100, 50 mM HEPES, 10% glycerol, 150 mM NaCl, and 1% sodium deoxycholate) supplemented with Phosphatase Inhibitor Cocktail 2 and 3 (Sigma Aldrich) and protease inhibitor cocktail (Sigma Aldrich). Then, proteins were separated on 4-12% SDS polyacrylamide gels (Bio-Rad Laboratories, MI, Italy) and then transferred electrophoretically to nitrocellulose membranes, using the Trans-Blot Turbo System (Bio-Rad Laboratories). The immunoreactive bands were visualized using the Enhanced Chemiluminescence (ECL) kit (GE Healthcare, Little Chalfont, UK) according to the recommendations of the manufacturer. The membrane was exposed to the ChemiDoc System (Bio-Rad Laboratories) and analyzed using Image Lab Software (Bio-Rad Laboratories). Antibodies specific for PPARγ, CEBPα, SREBP1c (Novus Biological, Centennial, Colorado, USA), Glut-4, SOD2, AMPK-α, phospho-AMPK-α (Thr172), HSL, phospho-HSL (Ser565), ATGL (Cell Signaling Technology, Danvers, Massachusetts, USA), Sirt1 and phospho-ATGL (Ser406) (Abcam Cambridge, UK), p21, cyclin D3, and vinculin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used.

Cells were lysed in a RIPA buffer, and protein concentrations were determined by using the Pierce BCA kit (Life Technologies) according to the manufacturer’s instructions. Equal protein concentrations (determined by Bradford assay) were loaded onto an SDS-PAGE gel. Proteins were then transferred to a PVDF membrane and probed with antibodies for Glut1 (Abcam, ab115730), CPT1-M (Alpha Diagnostic, CPT1M11-A), UCP1 (Abcam, ab10983), PPARγ (Santa Cruz Biotechnology, sc-7273), HSL (#18381), Phospho-HSL (Ser563: #4139, Ser565: #4137, Ser660: #4126), ATGL (#2439), FABP4 (AP2: #3544), GAPDH (#2118), and b-tubulin (#2146) (all from Cell Signaling), followed by incubation with appropriate secondary antibodies and visualization with enhanced chemiluminescence substrate.

Western blotting was performed as previously described^{27 (link)}. Briefly, proteins from adipose tissue, differentiated 3T3-L1 cells, and EVs were extracted using radioimmunoprecipitation assay protein lysis buffer (P1003; Beyotime Institute of Biotechnology, Shanghai, China) with freshly added 1% protease inhibitor cocktail and 1 mM phenylmethylsulphonyl fluoride. Each well was loaded with 50 μg protein and incubated overnight at 4 °C with primary antibodies. We purchased antibodies phospho-PKA substrate (catalog number 9624), phospho-HSL (catalog number 4126), and HSL (catalog number 4107) from Cell Signaling Technology; antibodies against UCP1(catalog number ab10983), tubulin (catalog number ab18207), CD9 (catalog number ab92726), TSG101 (catalog number 125011), and Hsp70 (catalog number 2787) from Abcam; and an antibody against Rab27A from Proteintech (Wuhan, China). We performed quantitative analyses of protein levels with ImageJ software.

Protein was extracted from frozen liver and epididymal white adipose tissues (eWAT). Nuclear protein was prepared from mouse livers as described previously (Alam et al., 2017 (link)). Protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Fisher Scientific, MA). Western blotting was performed as described previously (Liu et al., 2020 (link)). Primary antibodies (1:500–1:1,000) against Phospho-HSL, ATGL, PPAR-γ, CPT-1a, Nrf2, BAX, BCL-2, Adiponectin, Cleaved-Caspase3, pro-Caspase3 and CYP7A1 were from Cell Signaling Technology (MA, United States). β-Actin and Lamin B antibody were the reference of total protein and nuclear protein, respectively, and purchased from Abcam (Cambridge, MA, United States). Densitometric analysis was performed using UN-SAN-IT Gel (Silk Scientific, Orem, UT) software.