Ab169776

Extracts containing equal amounts of total protein (20 μg) were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% nonfat milk solution for 60 min, membranes were incubated with specific antibodies overnight at 4°C. The antibodies used were against the following antigens: LBP (anti-human LBP antibody; 1 : 1000 dilution, ab169776, Abcam Ltd., Cambridge, United Kingdom), LBP (anti-rat LBP antibody; 1 : 1000 dilution, sc-14666, Santa Cruz Biotechnology), p38 MAPK (1 : 2000 dilution, 8690, Cell Signaling Technology), phospho-p38 MAPK (1 : 2000 dilution, 9910, Cell Signaling Technology), phospho-p65 (1 : 2000 dilution, 9936, Cell Signaling Technology), FKN (1 : 1000 dilution, ab25088, Cell Signaling Technology), and beta-actin (1 : 2000 dilution, SC-47778, Santa Cruz Biotechnology). After primary antibody incubation, the membranes were incubated with horseradish peroxidase conjugation (9936, Cell Signaling Technology). Detection was accomplished by using an enhanced chemiluminescence detection system. The images of western blots were scanned by Quantity One software, and the original intensity of each specific band was quantified with freeware image analysis software, NIH Image (National Institute of Health, Bethesda MD, USA).

A Universal Magnetic Co-IP Kit (54002, Active Motif, Carlsbad, CA, USA) was used for Co-IP experiments. Three micrograms of LBP antibody (ab169776, Abcam Ltd., Cambridge, United Kingdom) was added to 500 μg of extracted proteins. The mixture was incubated at 4°C for 60 min with gentle mixing. Then, 25 μL of Magnetic Protein G Beads was added and incubated at 4°C overnight. The mixture was centrifuged at 4000 rpm for 30 seconds at 4°C. The supernatant was discarded, and the Co-IP products were washed four times with PBS. After the final wash, the precipitates were resuspended in 20 μL of sample buffer for western blotting assay. Three micrograms of rabbit IgG (A7016, Beyotime Biotechnology, Nantong, China) and no antibody were used instead of anti-LBP antibody for negative control and blank control, respectively.