Pparα antibody

The total cellular or liver tissue protein was obtained by lysing the cells with RIPA lysate (Sigma, V900854), and the protein concentration was determined using a BCA protein concentration assay kit (Sigma, FP0010). Equal amounts of protein were electrophoresed on a 10% Bis-Tris gel at 120 V for 1 h, the protein was transferred to the PVDF membrane at 350 mA for 70 minutes, and the PVDF membrane was blocked with 5% BSA in TBST buffer for 1 h. The primary antibody was incubated by gentle shaking at 4°C overnight, and the secondary antibody was incubated for 1 h at room temperature. ECL hypersensitive luminescent solution (Thermo, 32132) was used for color reaction, and gray scale was detected by image laboratory software (Bio-Rad) to quantitatively analyze protein expression. The antibodies used included HNF1α antibody (Abcam, ab96777), IRS-1 antibody (CST, #2382), phospho-IRS-1 antibody (CST, #2385), AKT antibody (CST, #9272), phospho-Akt antibody (CST, #4060), SOCS3 antibody (CST, #2932), STAT3 antibody (CST, #9139), phospho-STAT3 (CST, #9134), SREBP1 antibody (Abcam, ab191857), and PPARα antibody (Abcam, ab8934). The GAPDH protein antibody (Abcam, ab8245) was selected as an internal reference.

Hearts from mice treated with Wy-14,643 (250 mg/kg body weight) for 24 h and H9c2 cells treated with Wy-14,643 (100 μM) for 48 h were used for chromatin immunoprecipitation (ChIP) assay using ab500 ChIP kit (Abcam, Cambridge, MA USA). Thirty milligrams of chopped heart tissue and 1 × 10⁶ H9c2 cells were fixed with 1% formaldehyde and processed according to manufacturer’s protocol. Briefly, fixed tissue or cells were lysed and sonicated using Fisher 300 Sonic Dismembrator (Fisher Scientific, Hampton, NH, USA) to obtain optimal DNA fragments ranging from 200 to 1000 bp. Chromatin was immunoprecipitated with PPARα antibody (ab2779, Abcam, Cambridge, MA USA). or mock IgG followed by reverse crosslinking and DNA purification. PCR was conducted using primers spanning each of the three PPREs on Pde1C promoter (Table S1). The PCR product after 35 cycles was run on 1% agarose gel. To quantify the promoter enrichment, 1 μL of purified DNA was analyzed by RT-qPCR, using similar conditions used for PCR. Triplicate C_t numbers from sample and mock were used to determine fold enrichment of promoter fragments as compared to mock.