Anti phsl s563

Human adipocytes were isolated from abdominal subcutaneous adipose tissue by collagenase digestion. Cells were resuspended in KRB-HEPES containing 25 mM HEPES (pH 7.4), 2 mM glucose, 1% (w/v) BSA and 200 nM adenosine and then stimulated with insulin (10 nM, 10 min) or isoprenaline (30 nM, 30 min). After stimulation, cells were washed in KRB-HEPES (without BSA), and lysed in 50 mM TRIS-HCl (pH 7.5), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM dithiothreitol, 1% (w/v) NP-40 and complete protease inhibitor cocktail (one tablet/50 ml). Lysates were centrifuged at 13,000 × g for 15 min (4 °C) and the protein concentration in the supernatant was determined by the Bradford assay. Lysates (5 µg) were then subjected to Western blot analysis, as described previously^{63 (link)}. The following primary antibodies were used: anti-pHSL S563 (Cell Signaling Technology (CST) #4139, 1:1000), anti-HSL (CST #4107, 1:1000), anti-pPerlipin S522 (Vala Science #4856, 1:1000), anti-Perlipin (CST #9349, 1:5000), anti-pPKB S473 (Thermo Fisher #44621 G, 1:2000), anti-PKB (CST #9272, 1:1000) and anti-β-actin (Sigma #A5441, 1:2000).

Dulbecco’s modified Eagle medium (DMEM), Dulbecco’s phosphate-buffered saline (DPBS), fetal bovine serum (FBS), and 0.25% trypsin were purchased from Gibco (Rockville, MD, USA). Penicillin-streptomycin solution was purchased from Hyclone (Provo, UT, USA). Insulin, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), and MTT were obtained from Sigma-Aldrich (St. Louis, MO, USA). 3T3-L1 preadipocytes were purchased from ATCC (Manassas, VA, USA). Bilobalide was obtained from the National Institutes for Food and Drug Control (Beijing, China). Primary antibodies specific for anti-pACC1 (S79), anti-ACC1, anti-FASN, anti-perilipin A, anti-ATGL, anti-C/EBPα, anti-PPARγ, anti-HSL, anti-pHSL (S563), anti-GLUT-4, anti-CPT-1α, anti-AMPK, and anti-pAMPK (T172) were acquired from Cell Signaling Technology (Danvers, MD, USA). Anti-SREBP-1c was acquired from Abcam (Cambridge, UK). Anti-β-Actin and goat anti-mouse and goat anti-rabbit IgG secondary antibodies were obtained from Boster Biological Technology (Pleasanton, CA, USA). The AMPK assay kit was purchased from Cell Signaling Technology.

AZD1208 was purchased from Selleckchem (Houston, TX, USA). Primary antibodies for anti‐C/EBP‐α, anti‐PPAR‐γ, anti‐phospho (p)‐STAT‐3 (Y705), anti‐STAT‐3, anti‐Pim‐1, anti‐Pim‐2, anti‐Pim‐3, anti‐p‐Bad (S112), anti‐Bad as well as secondary goat anti‐rabbit and goat antimouse IgG antibodies were purchased from Santa Cruz Biotechnology (Delaware, CA, USA). Primary antibodies specific for anti‐p‐AMPK (T172), anti‐AMPK, anti‐p‐ACC (S79), anti‐ACC, anti‐p‐LKB‐1 (S428), anti‐LKB‐1, S6, anti‐p‐HSL (S563) and anti‐p‐HSL (S660) were acquired from Cell Signaling Technology (Danvers, MA, USA). Anti‐HSL antibody was obtained from Cayman chemical (Ann Arbor, MI, USA). Anti‐perilipin A antibody was purchased from Bio Vision (Milpitas, CA, USA). Anti‐FAS antibody was purchased from BD Bioscience (San Jose, CA, USA). Anti‐actin antibody, 3‐isobutyl‐1‐methylxanthine (IBMX), dexamethasone and insulin were purchased from Sigma (St. Louis, MO, USA).