P p38 antibody

Frozen left ventricular tissues (100 mg) were grounded and lysed with RIPA lysis buffer. The lysates were boiled for 30 minutes and centrifuged at 12,000 rpm for 6 minutes. The proteins were loaded onto SDS-polyacrylamide gels and underwent electrophoresis. The proteins were then electro-blotted onto the PVDF membranes (Millipore, USA). Subsequently, the membranes were separately incubated with rabbit polyclonal to transforming growth factor-β1 (TGF-β1) antibody (Abcam, USA, 1:5000 dilution), nuclear factor kappa B (NF-κB) P65 antibody (Abcam, USA, 1:1000 dilution), p38 antibody (Abcam, USA, 1:1000 dilution), P-p38 antibody (Abcam, USA, 1:1000 dilution), MMP-9 antibody (Abcam, USA, 1:500 dilution), p22phox antibody(1:1000 dilution), gp91phox antibody(1:500 dilution), rac1 antibody(1:1000 dilution), and were incubated with appropriate peroxidase-conjugated secondary antibodies. Reactive blots were visualized by using Western Lightning^TM Chemiluminescence Reagent (Millipore) and quantified using densitometry.

Protein extracts of rat spinal cord tissue L4-6 were added to a homogenizer. The supernatant was obtained by centrifugation after thorough trituration. SDS protein loading buffer was added to the supernatant to extract protein samples. Target proteins of different molecular weights were obtained by electrophoresis. After being attached to the NC protein membrane, the membrane was transferred and blocked. Membranes were washed 3 times with TBS before being transferred to primary antibodies and incubated overnight at 4°C. The p38 antibody (Abcam, USA), p-p38 antibody (Abcam, USA), OX42 antibody (Abcam, USA), ASK1 (Abbkine, China), MKK3 (Abbkine, China), and β-actin antibody (Abcam, USA) were added, respectively. After washing, the membranes were incubated in secondary antibody and rinsed with TBS solution 3 times. The ECL working droplets were added to the surface of the NC membrane, and then the membrane was placed in an FCM gel imager for exposure and photography.

Anti-TLR4, p38 and p-p38 antibodies were from Abcam (Cambridge, USA). Anti-IL-6 and anti-MCP-1 were purchased from Cell Signaling Technology, Inc. (Danvers, USA). Anti-β-actin antibodies were obtained from Novus Biologicals, Inc. (Littleton, CO, USA). The shRNAs were synthesized by Cyagen Bioscience Inc. (Guangzhou, China). Anti-β-actin antibodies were obtained from Proteintech (Chicago, IL, USA). Secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA), and Dylight 594 conjugated secondary antibodies were from Pierce, Thermo Fisher Scientific Inc. (Rockford, IL, USA). The p38 inhibitor SB203580 was from Selleck (Houston, TX, USA). LPS were purchased from Sigma Chemical Company (St Louis, MO, USA).