Pgc1α antibody

For confocal microscopic imaging, cancer cells were cultured on glass coverslips. Mitochondrial staining was carried out using 200 nM MitoTracker Red CMXRos (M7512, ThermoFisher) at 37 °C for 30 min. Afterwards, the cells were fixed for 10 min in prewarmed 4% paraformaldehyde, and then washed with pre-warmed phosphate-buffered saline (PBS). Mitochondrial ROS production was visualized by MitoSox RED (M36008, ThermoFisher; 37 °C, 30 min), while PGC1α staining using PGC1α antibody (Millipore, ST1202). The coverslips were mounted using the ProLong Diamond Antifade Mountant containing 4′6-diamidino-2-phenylindole (DAPI) (Thermo Fisher, Cat#: P36962) for nuclear staining (22 °C, 30 min). Images were acquired with a Leica TCS SP2 AOBS (Acoustico Optical Beam Splitter) inverted laser scanning confocal microscope equipped with a 63 × water immersion objective (HCX PL APO 63.0 × 1.20 water corrected). DAPI and MitoTracker Red CMXRos were excited with Ultraviolet or 359 nm and 579 nm lasers, respectively. Images processing were carried out with ImageJ software (imagej.nih.gov).

Liver proteins were quantified by western blot analysis as follows using BioRad, (Hercules, CA) Criterion and ChemiDoc systems following manufacturers’ instructions. PGC1α antibody was purchased from EMD Millipore, Billerica, MA, Cat# ST1202. All other antibodies purchased from Santa Cruz Biotechnology, Santa Cruz, CA were as follows: FOXO1Ser256 Cat# sc-101681; G6Pase-α Cat# sc-27198; PEPCK Cat# sc-32879; PPARα Cat# sc-9000; SREBP-1 Cat# sc-13551; mTORC Cat# sc-8319; PPARγ Cat# sc-7273; PGC1β Cat# sc-67286, NFκB p65 Cat# sc-8008; IKKαβ Cat# sc-7607; SOCS3 Cat# sc-9023; JNK Cat# sc-571; Actin Cat# sc-47778. Criterion gradient tris-glycine precast gels, secondary antibodies, PVDF blotting membranes, molecular weight markers, and Enhanced Chemiluminescence (ECL) reagents were also purchased from BioRad. Samples from 8 SHR Vehicle, 8 SHR Bromocriptine treated rats, and 6 Wistar wild type controls were loaded onto the same 26 well Criterion gel along with the molecular weight markers; band intensity was compared only within the samples loaded onto the same gel. A housekeeping protein (Actin) was concurrently quantified on all gels, and the test protein amount was normalized to Actin in the Western blot analysis. Protein bands were quantified with BioRad ImageLab 4.1 software.