Rabbit anti p38 mitogen activated protein kinase p38 mapk

Synthetic LXR ligand 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)-(2,2-diphenylethyl) amino] propyloxy] phenylacetic acid hydrochloride (GW3965) was kindly donated by Jon Collins (GlaxoSmithKline, Research Triangle Park, NC). Dihydroethidium (DHE) and TRIzol Reagent were from Life Technologies (Carlsbad, CA). Mouse monoclonal antibody against LXRα (ab41902) and rabbit polyclonal antibody against LXRβ (ab28479) were from Abcam (Cambridge, UK); rabbit anti-mouse nitrotyrosine antibody (06-284) was from Millipore (Billerica, MA); rabbit anti-cleaved caspase-3 (5A1E, #9664), rabbit anti-nuclear factor kappa-light-chain-enhancer of activated B cell p65 (NF-κB p65, C22B4; #4764), rabbit anti-Akt (#9272), rabbit anti-phospho-Akt (D9E, Ser473, #4060), rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK, #9212), rabbit anti-phospho-p38 MAPK (D3F9, Thr180/Tyr182, #4511), rabbit anti-c-Jun N-terminal kinase (JNK, 56G8, #9258), rabbit anti-phospho-JNK (81E11, Thr183/Tyr185, #4668), rabbit anti-Histone H3 (#9715) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 14C10, #2118) were from Cell Signaling Technology (Beverly, MA). IRDye 800CW goat anti-mouse (926-32210) and anti-rabbit IgG (926-32211) secondary antibodies were from LI-COR Biosciences (Lincoln, NE).

The ganglia or cells were homogenized with radio immunoprecipitation assay lysis (Applygen, Beijing, China, Cat# C1053+, 2017). The concentration of protein supernatant was measured by the Lowry method, then diluted with protein loading buffer (Transgen Biotech, China) and bathed in boiling water for 5 min. Aliquots of protein were separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membrane (Millipore, United States). The membrane was blocked for 2 h, then incubated with rabbit anti-P2Y12 (1:2,000 dilutions, Alomone Labs, Israel), rabbit anti-p38 mitogen-activated protein kinase (p38 MAPK), rabbit anti-phosphorylated-p38 (P-p38 MAPK) (1:1,000 dilutions, Cell Signaling Technology, United States), and mouse monoclonal anti-β-actin (1:800 dilutions, ZSGB-Bio) antibodies at 4°C overnight. After being washed, the membrane was incubated with the secondary antibody (anti-mouse IgG, goat anti-rabbit IgG, 1:2,000, ZSGB-Bio). The Bio-Rad system was used for labeled proteins visualized. Image-J was applied to quantify the results (Zhao et al., 2019 (link)).