Anti p p38 antibody

Renal cortex and cultured podocyte were lysed by lysis buffer on ice. Equal amounts of protein (20 μg per lane) were subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked by 5% nonfat dry milk in PBS + 0,05% Tween 20. After that, the membranes were incubated with primary antibodies at 4°C overnight. After washing with PBS, peroxidase secondary antibody was added and incubated for 1 h at room temperature. Antibodies are the following: anti-Nox4 antibody (Abcam, UK), anti-P38 antibody (Abcam, UK), anti-P-P38 antibody (Abcam, UK), anti-Bax antibody (Abcam, UK), anti-Bcl-2 antibody (Abcam, UK), and anti-cleaved caspase-3 antibody (Abcam, UK). The blots were visualized with LumiGLO reagent and peroxide, followed by exposure to X-ray film. Western Blot analyses were performed at least in triplicate.

The cerebral cortex tissues with blood clots or cultured cells were collected and lysed in 20 mM Tris (Beyotime, China). Western blotting was conducted following the standard protocol. The following primary antibodies were used: anti-induced nitric-oxide synthase (iNOS) antibody, anti-adenosine A3 receptor antibody, anti-liver arginase antibody (Arg-1), anti-cleaved caspase 3 antibody, anti-IL-4 antibody, anti-IL-13 antibody, anti-p-P38 antibody (Thr180/Tyr182), anti-p-STAT6 antibody (Tyr641), anti-p-JAK1 antibody (Tyr1022/Tyr1023), anti-p-STAT1 antibody (Tyr701), and anti-p-NF-κB p65 antibody (Ser276) (Abcam, USA). Anti-tubulin rabbit antibody and goat anti-rabbit and rabbit anti-goat secondary antibodies were used (Beyotime, China). The blotted protein bands were quantified by Image J software (National Institutes of Health, USA). The density values are shown as the target protein OD/β-tubulin OD ratio.

The expression of phosphorylated p38 (p-p38) in T lymphocytes was analyzed by western blot. For protein extraction, T lymphocytes from the recipient mice were treated with RIPA lysis buffer (Beyotime), and the concentration of protein was assessed by using a BCA protein assay kit (Beyotime). Total protein was separated with 12% SDS–PAGE and transferred onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was then blocked with tris-buffered saline tween-20 (TBST) containing 5% nonfat milk for 1 h at RT, and then incubated with the following primary antibodies: anti-p-p38 antibody (Abcam, 1:200), anti-β-actin antibody (Abcam, 1:3000) for 1 h at RT. The membrane was then incubated with HRP-bounded secondary antibodies for 1 h at RT and the proteins were visualized with an ECL Plus Western Blotting Substrate (Thermo Fisher). β-actin served as control to evaluate the relative expression.