Rabbit anti pparγ

Proteins were extracted from hypothalamus and the samples in SDS Loading Buffer were heated (100°C, 5 min), subjected to SDS-PAGE, transferred to PVDF or nitrocellulose membranes, and blocked (4°C, overnight) in PBST (PBS with 0.05% Tween 20) containing 5% nonfat dry milk or 5% BSA. Blots were incubated with a primary antibody in blocking buffer (overnight, 4°C) and then with a second antibody (1 : 1000~2000 dilution, 1 hr, RT). Signals were detected using SuperSignal ^® West Femto Maximum Sensitivity Substrate. Immunodetection of endogenous GAPDH was utilized to indicate that equivalent amounts of protein were present in samples added to the SDS PAGE (wells/lanes·μg/L).
Rabbit anti-GAPDH (Cell Signaling, Cat#2118, 1 : 2000 dilution for Western blot); rabbit anti-PPARγ for Western blot (Cell Signaling, Cat#2443, 1 : 1000 dilution for Western blot); rabbit anti-Stat3 (phospho Y705) for Western blot (Abcam, ab76315, 1 : 1000 dilution for Western blot); and rabbit anti-leptin (Abcam, ab3583, 1 : 1000 dilution for Western blot) were used.

Immunoblotting was performed as previously described [44 (link)]. The cells were grown in a 12-well plate, and the experiments were repeated at least three times. Cell lysates were prepared using a cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing 1 μM PMSF (Roche, Basel, Switzerland). The protein levels of the obtained lysates were quantified using BCA Protein Assay reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cell lysates were diluted in a 5× sodium dodecyl sulfate (SDS) sample buffer and then separated on 10% SDS-PAGE gel. The separated proteins were transferred onto polyvinylidene fluoride membranes (Invitrogen, Carlsbad, CA, USA), which were then incubated with appropriate primary and secondary antibodies. Specific proteins were detected using rabbit anti-PPARγ (Cell Signaling Technology), mouse anti-SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-CREB (Cell Signaling Technology), rabbit anti-phospho-CREB (ser133, Cell Signaling Technology), chicken anti-ADRB2 (Abcam, Cambridge, MA, USA), and mouse anti-GAPDH (Santa Cruz Biotechnology). Protein bands were detected using horseradish peroxidase-conjugated anti-chicken IgY (Abcam), anti-rabbit IgG (AbFrontier, Seoul, Korea), and anti-mouse IgG (AbFrontier) antibodies, and enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).

T0070907 and Cell Proliferation ELISA, BrdU(colorimetric) were purchased from Sigma Inc. (St. Louis, MO, USA). Pioditazone hydrochloride (HPLC ≥ 98%) was purchased from Dalian Meilun Biotech Co., Ltd (Dalian, China). Rabbit anti-PPAR-γ, anti-β-catenin, anti-c-Myc and rabbit anti-Cyclin D1 monoclonal antibody were purchased from Cell Signaling (Danvers, MA, USA) and Rabbit anti-β-actin monoclonal antibody was purchased from Bioworld (Shanghai, China). Secondary antibody for goat anti-rabbit immunoglobulin (IgG) horse radish peroxidase (HRP) was purchased from Beijing Zhongshan Biotechnology Corporation (Beijing, China). PPAR-γ and β-actin primers were produced from Shanghai Sangon Biological and Technological Company (Shanghai, China).