Akt mouse monoclonal antibody

WB was performed as previously described^{27 (link)}. The following primary antibodies were used: TC2N rabbit polyclonal antibody (1:500; Abcam), p85 mouse monoclonal antibody (1:1000; Santa Cruz), p-p85 Tyr508 goat polyclonal antibody (1:1000; Santa Cruz), p-p55γ Tyr199 rabbit polyclonal antibody (1:1000; Bioss), p55γ mouse monoclonal antibody (1:1000; Santa Cruz), AKT mouse monoclonal antibody (1:1000; Santa Cruz), p-AKT Ser473 mouse monoclonal antibody (1:1000; Santa Cruz) GSK3B mouse monoclonal antibody (1:1000; Santa Cruz), p-GSK3B mouse monoclonal antibody (1:1000; Santa Cruz), p-MYC mouse monoclonal antibody (1:1000; Santa Cruz), p-BAD mouse monoclonal antibody (1:1000; Santa Cruz), Caspase-3 mouse monoclonal antibody (1:1000; Santa Cruz), cleaved-Caspase-3 rabbit monoclonal antibody (1:1000; Cell Signaling Technology), ALK mouse monoclonal antibody (1:1000; Santa Cruz), EBP1 mouse monoclonal antibody (1:1000; Santa Cruz) and ACTIN monoclonal antibody (1:2000; Sigma). ACTIN served as a loading control.

Dulbecco's Modified Eagle Medium, and heat-inactivated fetal bovine serum (FBS) were purchased from Gibco Life Technologies (Grand Island, NY, USA). Antibiotic antimycotic solution (Penicillin: Streptomycin: Neomycin solution), all-trans retinoic acid, corn oil, bovine serum albumin, O.C.T. tissue-freezing medium, Oil red O, paraffin, PBS, citrate buffer, hydrogen peroxide, 3,3′ diaminobenzidine, PMSF, Protease Inhibitor Cocktail, and NP-40 were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Glucose concentration was measured using a glucometer (Accu-chek Performa, Roche, Mannheim, Germany). Antibodies to the following targets were used: anti-UCP-1 polyclonal antibody was purchased from Cloud-Clone Corp. ((CCC), USA), AKT mouse monoclonal antibody was purchased from Santa Cruz Biotechnologies (USA) and rabbit anti- IgG–horseradish peroxidase secondary antibodies were purchased from Abcam (Cambridge, UK). Plastics and EDTA/aprotinin vacutainer tubes were from Becton Dickinson (BD Biosciences, Franklin Lakes, NJ, USA).

Proteins were extracted from PVAT homogenized in a Tissue Lyser (Qiagen, Germany) using a lysis buffer (0.01 M PBS; 0.001 M phenylmethylsulfonyl fluoride (PMSF); Protease Inhibitor Cocktail (Sigma-Aldrich Chemical Co., St. Louis, MO, USA); 1% NP-40 (v/v). The total protein concentration was determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, USA). Adipose tissue explants lysate protein was separated by the SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was immunoblotted with an anti-UCP-1 polyclonal antibody (Cloud-Clone Corp. (CCC), USA) and AKT mouse monoclonal antibody (Santa Cruz Biotechnologies, USA) overnight followed by rabbit anti- IgG–horseradish peroxidase secondary antibodies (Abcam, Cambridge, UK). Protein bands were detected with a light-emitting nonradioactive method using ECL reagent prepared in the laboratory^{23 (link)}. The membranes were then subjected to autoluminography for 1 to 5 min.