Rabbit anti phospho p38 mapk antibody

The S1PR1 specific inhibitor W146 (sc 296700) was purchased from Santa Cruz Biotechnology (CA, USA). The drug-dissolving agent dimethyl sulfoxide (DMSO), S1PR1 specific agonist SEW2871 (S3944), ERK/MAPK-specific inhibitor PD98059 (P215), p38/MAPK specific inhibitor SB202190 (S7067), and JNK/MAPK-specific inhibitor SP600125 (S5567) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Rabbit anti-S1PR1 (EDG-1) antibody (BS2593) was purchased from Bioworld (MN, USA). Rabbit anti-chicken NLRP3 polyclonal antibody was gifted by Zhangyong Ning (South China Agricultural University). Rabbit anti-GAPDH antibody (ab181602) was purchased from Abcam (Cambridge, UK). Rabbit anti-phospho-p44/42 MAPK (Erk1/2) antibody (#9101), rabbit anti-p44/42 MAPK (Erk1/2) antibody (#4695), rabbit anti-phospho-p38/MAPK antibody (#4511), and rabbit anti-p38/MAPK antibody (#8690) were purchased from Cell Signaling Technology (MA, USA). Rabbit anti-phospho-JNK1/MAPK antibody (bs-17591R) was purchased from Bioss Biotechnology Co., Ltd. (Beijing, China). Rabbit anti-JNK1/MAPK antibodies (ab199380) were purchased from Abcam. Rabbit anti-NDV-NP polyclonal antibodies were prepared and stored in our laboratory.

The following commercial antibodies from Cell Signaling Technology were used : rabbit anti-p38 MAPK antibody (catalog no. 8690, 1:4000 dilution); rabbit anti-phospho-p38 MAPK antibody (catalog no. 4511, 1:2000 dilution); mouse anti-p44/42 MAPK (Erk1/2) antibody (catalog no. 4696, 1:2000 dilution); rabbit anti-phospho-p44/42 MAPK (Erk1/2) antibody (catalog no. 4370, 1:4000 dilution); mouse anti-β-actin antibody (catalog no. 3700, 1:4000 dilution); and peroxidase-conjugated goat anti-rabbit IgG (catalog no. 7074, 1:4000 dilution). Peroxidase-conjugated sheep anti-mouse IgG antibody was obtained from Cytiva (Tokyo, Japan; catalog no. NA931, 1:2000 dilution). Peroxidase-conjugated donkey anti-rat IgG antibody was obtained from Jackson ImmunoResearch (West Grove, PA, USA; catalog no. 712-035-150, 1:5000 dilution). Rat anti-mCherry antibody was obtained from Thermo Fisher Scientific (catalog no. M11217, 1:1000 dilution).

The samples of brain tissues were homogenized in RIPA lysis buffer with protease or phosphatase inhibitor (Roche, Basel, Switzerland) and then centrifuged (12000 rpm, 15 min, 4°C) to obtain supernatants. The total protein concentration of the supernatants was determined using the BCA assay kit (Pierce). Samples were loaded and separated on the SDS–PAGE gel (40 μg per well). The protein was transferred to the PVDF membrane (Millipore, MA, United States), which was incubated overnight with primary antibodies at 4°C and then probed with the corresponding secondary antibody. The primary antibodies were as follows: rabbit anti-AT₁R antibody (1:800 dilution, Abcam, MA, United States) (Czikora et al., 2015 (link); Wang et al., 2018 (link)), rabbit anti-SAPK/JNK antibody, mouse anti-phospho-SAPK/JNK antibody, mouse anti-ERK1/2 antibody, rabbit anti-phospho-ERK1/2 antibody, rabbit anti-p38MAPK antibody, rabbit anti-phospho-p38MAPK antibody and rabbit anti-β-tublin antibody (1:1000 dilution, Cell Signaling Technology, Danvers, MA, United States). The membranes were detected by an ECL-Plus detection kit (Tiangen, Beijing, China), and scanned using Image Quant LAS 4000 (GE Healthcare Life Sciences, CT, United States). The images were quantified using the ImageJ densitometry system.