Rabbit anti pparγ polyclonal antibody

PPARγ, p38, phospho-p38 protein in BMSCs under different culture conditions were measured. Proteins were harvested with whole cell lysis assay kit (Keygen, Nanjing, China). Cells were washed in cold PBS, followed by addition of 1ml lysis buffer, then agitated strongly for 4 seconds at 4°C, and placed on the ice for 4 min. This was repeated five times. Cell lysates acquired were centrifuged at 12,000 g for 5 min at 4°C, then the supernatant protein was harvested and total protein concentration was determined using BCA protein assay kit (Keygen, Nanjing, China). After separating the protein samples on 10% SDS-PAGE gels, transferring to polyvinylidenedifluoride membranes (Millipore, USA), these membranes were immunoblotted by rabbit anti-PPARγ polyclonal antibody (Abcam, Cambridge, UK)) at 4°C overnight and incubated with secondary antibody (Abcam, Cambridge, UK)) at room temperature for 1h. PPARγ, p38 and phospho-p38 were visualized using enhanced chemiluminescence reagents (Millipore, USA). The immunoblots were quantified with software Image J (National Institute of Health, Maryland, USA).

Cells with or without LMHFV application and those with or without SB203580, were seeded on millicell EZ 8-well slide. After adherence, they were washed in PBS, fixed in 4% buffered (0.01mol/L PBS, pH 7.2, 0.1%DEPC) paraformaldehyde for 30 min at room temperature, and blocked by 0.5% bovine serum albumin (BSA) for 15 min. After overnight incubation with rabbit anti-PPARγ polyclonal antibody (1:60) (Abcam, Cambridge, UK)[15 ] at 4°C, cells were subsequently incubated with secondary antibody conjugated to Rhodamine (Hebei Bio-high Technology Deve co., Hebei, China) for 20 min at 37°C. After rinsing in PBS, cells were observed under the Olympus IX 71 inversed fluorescent microscope (Olympus, Tokyo, Japan). Images were captured by Image-Pro Plus version 6.0 (Media Cybernetics) and analyzed by Image J software (National Institute of Health, Maryland, USA).