Rabbit monoclonal anti pparα antibody

Western blot analysis was performed as described previously [16 (link)]. Briefly, 50μg of each protein sample was separated on 10% SDS-PAGE and transferred onto PVDF membrane. The membrane was incubated overnight with a primary antibody against each target protein (mouse monoclonal anti-apoA5 antibody, Santa Cruz; or rabbit monoclonal anti-PPARα antibody, Abcam; mouse monoclonal anti-LDL receptor antibody, Progen Biotechnik) at 4°C overnight. After incubation with an HRP-conjugated secondary antibody, immunoreactive bands were visualized using the enhanced chemiluminescence detection system. Data were quantified by densitometry after scanning, using the TINA software (Raytest, Germany). The results of the target protein were presented relative to GAPDH expression.

Canine LAA tissues were lysed with RIPA Lysis Buffer (ASPEN, USA) supplemented with complete protease and phosphatase inhibitor cocktail (ASPEN, USA). We used Bicinchoninic acid (BCA) assay (ASPEN, USA) to estimated protein concentration after centrifugation at 13000 rpm for 5 min. Proteins were separated on SDS-polyacrylamide gels and transferred to PVDF membranes. Then, the membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit monoclonal anti-GAPDH antibody (diluted 1:10000, Abcam), rabbit monoclonal anti- p-AMPKα1 antibody (diluted 1:1000, Abcam), rabbit monoclonal anti- PGC-1α antibody (diluted 1:500, Abcam), rabbit monoclonal anti-PPARα antibody (diluted 1:1500, Abcam), rabbit monoclonal anti-FAT antibody (diluted 1:500, Bioss), rabbit monoclonal anti-VLCAD antibody (diluted 1:1000, Abcam) and then with secondary HRP-goat anti-rabbit antibody (diluted 1:10000, ASPEN) for 30 min at room temperature (RT). For loading controls, the membranes were stripped with stripping buffer (ASPEN, USA) for 10 min at room temperature. Antibody binding was detected with the ECL detection reagent (ASPEN, USA). At last, we quantified bands with AlphaEaseFC Software and data are shown as the ratio of total protein to GAPDH normalized to control.