P hsl

Tissues were homogenized in RIPA buffer (150 mM NaCl, 0.1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) supplemented with protease inhibitor (cOmplete Mini, Roche). The homogenate was centrifuged at 16,000 x g for 10 min at 4°C. The supernatant was assayed for protein concentration using a BCA assay. 15 μg of protein was resolved on a 10% SDS-PAGE gel (Invitrogen). Immunoblotting was performed using the following antibodies; P-CREB (Cell Signaling #9198), CREB (Cell Signaling #9197), pHSL (Cell Signaling #4139), HSL (Cell Signaling #4107), Ucp1 (Abcam #ab10983), and actin (Santa Cruz # sc-1615).

Protein extraction and Western blotting were performed according to standard protocols. Briefly, snap-frozen adipose tissue or cell samples were homogenized in ice-cold RIPA buffer (50 mM Tris–HCl pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA) containing 1 mM sodium orthovanadate, 1 mM sodium fluoride, and proteinase inhibitor cocktail (Roche), and centrifuged at 13,000×g for 15 min to collect supernatants. Supernatants (30 μg protein) were used for reducing SDS PAGE and Western blotting. Primary antibodies against p-HSL, HSL, p-PKA substrates, p-Perilipin, p-Smad2, β-actin, and α-tubulin (Cell Signaling, Sweden) were used at 1:2000 dilution. The levels of target proteins were quantified by the intensity of Western blot bands using ImageJ software (National Institutes of Health).

The lipid staining dye Oil Red O (ORO, catalog: O0625), thiazolyl blue tetrazolium bromide (MTT), cell differentiation reagents 3-isobutyl-1-methylxanthine (IBMX, catalog: I7018), dexamethasone (DM, catalog: D8893), and insulin (catalog: I5500) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Dulbecco’s Modified Eagle’s Medium (DMEM, catalog: 12430-054) and supplements including bovine serum (BS, catalog: 26170-043), fetal bovine serum (FBS, catalog: 16000-044), penicillin/streptomycin (P/S, catalog: 15140-122), and trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA, catalog: 15400-054) were purchased from GIBCO-BRL (Grand Island, NY, USA). Isopropanol (2-propanol, catalog: 5035-44) was purchased from Daejung Chemicals & Materials (Siheung-si, Korea). Formalin (catalog: 6936050380) for cell fixation was purchased from Junsei (Tyoko, Japan). Primary antibodies against adipogenesis-related proteins, including PPAR-γ (catalog: #2443), C/EBP-α (catalog: #2295) and FABP4 (catalog: #2120), and against thermogenic proteins such as PGC-1α (catalog: #2178) and p-HSL (catalog: #4139), were purchased from Cell Signaling Technology (Bedford, MA, USA). Secondary antibody, anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, catalog: sc-66163), and anti-SREBP-1 (catalog: sc-365513) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Cells and tissues were lysed in lysis buffer for 30 min, and protein quantification was performed using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Lysates containing 20 µg protein were electrophoresed on 8–12% SDS-PAGE gels and electrotransferred to membranes for ~2 h. The membranes were then immersed in 5% skim milk blocking buffer for 1 h and incubated with a primary antibody at 4 °C overnight. After several washes, secondary antibody and chemiluminescent detection reagents were sequentially applied. Antibodies targeting C/EBPα, PPARγ, p-AMPKα, AMPKα, CPT1, SREBP1, FAS, DGAT1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and ATGL, p-HSL, and HSL antibodies were from Cell Signaling Technology (Beverly, MA, USA).

The total protein was extracted with RIPA (Beyotime, Shanghai, China) and the concentration was mesured by BCA (Beyotime, Shanghai, China). An equal amount of protein was resolved by SDS–PAGE and transferred onto PVDF membrane. Then, the membrane was incubated with the primary antibody and the second antibody respectively. As shown in Table S3, primary antibodies against Atrogin-1 (muscle atrophy F-box protein) and MuRF1(muscle RING finger-1) were purchased from Santa Cruz Biotechnology (CA, USA). Primary antibodies against GAPDH and PDK4 (pyruvate dehydrogenase kinase 4) were acquired from Proteintech (IL, USA). Antibodies against P-PDH, FoxO1 (forkhead box O1), PDH, ATGL (adipose triglyceride lipase), HSL (hormone-sensitive lipase), P-HSL, and HSP90 were purchased from Cell Signaling Technology (MA, USA).

The ELISA kits for measuring the levels of TG, HDL-C, and LDL-C were purchased from Abcam (Cambridge, UK). The kit for measuring leptin level was purchased from Invitrogen (Carlsbad, CA, USA). The kit for measuring the AST level was purchased from BioVision Inc (Milpitas, CA, USA). The antibodies against C/EBPα, PPAR-γ, FABP4, FAS, p-HSL, p-ACC, and p-AMPKα were purchased from Cell Signaling Technology (Bedford, MA, USA). The antibody against SREBP-1c was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other chemicals and reagents used in this study were analytical grade.

eWAT was homogenized in RIPA buffer supplemented with protease and phosphatase inhibitors. Homogenates were then centrifuged at 14,000 rpm for 15 minutes, and supernatants were collected for protein analysis. Protein concentration in the supernatant was determined using BCA (bicinchoninic acid) protein assay. Western blot was performed to detect p-HSL, HSL, ATGL, PLIN, (Cell Signaling Technologies), and β-actin (Santa Cruz Biotechnology) according to the protocol described previously [17 (link)]. Blots were scanned using a Bio-Rad Imaging System (Image Lab™ Upgrade for ChemiDoc™ XRS+ System #170-8299). All specific bands were quantified with the Automated Digitizing System (Image Lab 4.1 programs). Results are representative of three independent experiments.