Anti rabbit igg hrp linked antibody cat no 7074

Epididymal adipose tissue samples were homogenized with a polytron tissue homogenizer in ice-cold lysis buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 100 mM NaF, 10 mM sodium pyrophosphate, and 1% Triton X-100) containing 5 mM iodoacetic acid and protease inhibitor (iNtRON Biotechnology, Inc., Seongnam, Korea), then solubilized at 4 °C for 40 min. Insoluble material was removed by centrifugation at 14,800× g for 10 min and the supernatant was collected and used for Western blot analyses. The protein contents of the lysates were measured using a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Western blot analyses were conducted as described previously [37 (link)]. The antibodies against p-AMPKα (T172) (Cat No. 2535), AMPKα (Cat No. 2532), and β-actin (Cat No. 3700) were obtained from Cell Signal Technology (Beverly, MA, USA). Anti-rabbit IgG HRP-linked antibody (Cat No. 7074) and anti-mouse IgG HRP-linked antibody (7076) were obtained from Cell Signal Technology. Blots were detected with Luminata^TM Forte Western HRP Substrate (Cat No. WBLUF0500, Millipore, Billerica, MA, USA). The relative protein abundances were determined using the ImageQuant^TM LAS 500 imaging system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Protein expression levels were normalized to that of β-actin.

Dulbecco’s modified Eagle’s medium (DMEM), phosphate buffered saline (PBS), and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, NY, USA). 96-Well plates were purchased from Corning Costar (Corning Inc., NY, USA). Aloe-emodin (Cat No. 110795–201710), rhein (Cat No. 110757–201607), physcion (Cat No. 110758–201616) (> 98% pure, free of endotoxin) were purchased from National Institutes for Food and Drug Control (Beijing, China), which were dissolved in DMSO and passed through a 0.22 μm filter (Pall Life Sciences, MI, USA) for sterilization and diluted with culture medium to final concentrations before treatment. In all experiments, the final DMSO concentration did not exceed 1‰ (v/v), so as not to affect cell growth. Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma Sigma-Aldrich Co. (St. Louis, MO, USA). Caspase-3 (8G10) Rabbit mAb (Cat No. 9665), Caspase-9 antibody (human specific) (Cat No. 9502), β-actin (13E5) Rabbit mAb (Cat No. 4970), anti-rabbit IgG, HRP-linked antibody (Cat No.7074) were obtained from Cell Signaling Technology (Danvers MA USA).