Erk1 2 mapk

Cells after nsPEF stimulation were collected at 0.5 h and lysed by RIPA lysis buffer (R0020, Solarbio). The western blotting was performed according to the manufacturer’s protocol [13 (link)]. Rabbit polyclonal antibodies against Phospho-P38 MAPK (4511, Cell Signaling), P38 MAPK (8690, Cell Signaling), ERK1/2 MAPK (4695, Cell Signaling), Phospho-ERK1/2 MAPK (4370P, Cell Signaling), JNK MAPK (9252, Cell Signaling), Phospho-JNK MAPK (4668, Cell Signaling), CREB (4820, Cell Signaling), Phospho-CREB (9198, Cell Signaling), STAT3 (4904, Cell Signaling), Phospho-STAT3 (9145, Cell Signaling), β-catenin (sc-7199, Santa Cruz Biotechnology), and β-actin (13E5, Cell Signaling) were utilized to detect the targeted proteins, followed by incubation with secondary HRP-linked antibody of anti-rabbit IgG (Cell Signaling). The complex of the antigen and the antibody was detected with TANON 1600 Gel Imaging System, and the expression level of protein is analyzed with Tanon Gis. Statistical significance was marked with different letters (P < 0.05).

Heart samples were homogenated by lysis buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na₃VO₄, 1 µg/mL aprotinin leupeptin and pepstatin, 1 mM Phenylmethylsulfonyl fluoride (PMSF)] and then centrifuged at 12 000 rpm for 15 min at 4°C was collected, and the protein concentration was measured using the Bicinchoninic Acid (BCA) protein assay (Beyotime, China). Approximately 50 µg protein was loaded onto 10% or 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline and incubated with different primary antibodies at 4°C overnight. The following antibodies were used in this study: Bcl-2, Bax, p67^phox, P38 MAPK and phospho-P38 MAPK(Thr180/Tyr182), Jun NH2-terminal kinase (JNK) and phosphor-JNK (Thr183/Tyr185), ERK1/2MAPK and phosphor-ERK1/2 MAPK(Thr202/Tyr204) (Cell Signalling Technology, USA) used at a 1∶1,000 dilution and gp91^phox, p47^phox, p67^phox (Santa Cruz Biotechnology, CA) used at a 1∶200 dilution. The membrane was treated with horseradish peroxidase-conjugated secondary antibody for 1 h at 37°C. Blots were developed by ECL kit (Pierce Biosciences, USA). The intensity of protein band was semi-quantitative measured by image analysis software (GeneTools from SynGene).

Protein from the kidney cortex or HK-2 cells was extracted and western blot analysis was performed as described previously47 (link). Antibodies used in this study included: collagen I, collagen IV (Southern Biotech), CTGF, Phospho-p70s6k, p70s6k, β-actin (Santa Cruz), CD32b (R&D), Phospho- NF-κB/p65, NF-κB/p65, phospho-Smad3, Smad3, Phospho-mTOR (Ser2448), mTOR, Phospho-ERK1/2 MAPK, ERK1/2 MAPK, Phospho-p38 MAPK, p38 MAPK (Cell Signaling), and LI-COR IRDye 800-labelled secondary antibodies (Rockland Immuno-chemicals). The detection of specific signals was performed by using the Odyssey infrared image system (LI-COR Biosciences, Lincoln, NE, USA) and quantified by Image J software (National Institutes of Health). The ratio for the protein detected was normalized against β-actin and expressed as the mean ± S.E.M.