Rabbit anti p44 42 mapk erk1 2 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.

dHL-60 cells were incubated with IgG (200 μg/mL), RTX (200 μg/mL), Fc fragments (70 μg/mL), LPS (1 μg/mL), or PMA (100 nM) for 5, 10, and 20 min in complete culture medium at 37 °C. The cells were then lysed in RIPA buffer supplemented with protease inhibitor cocktail (Roche Applied Science, Penzberg, Germany). The lysates were then electrophoresed in 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). The membranes were probed with rabbit anti-p44/42 MAPK (Erk1/2) antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-p44/42 MAPK (Erk1/2) antibody (Thr202/Tyr204) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-Syk (Tyr525/526) antibody (Cell Signaling Technology, Danvers, MA, USA), mouse anti-Syk antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-SHP-1 (Tyr564) antibody (Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-SHP-1 antibody (Cell Signaling Technology, Danvers, MA, USA), while mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Sigma-Aldrich, St. Louis, MO, USA) was used as internal control. After washes, the conjugates were then stained with horseradish peroxidase-conjugated secondary antibodies. Image densitometry was analyzed by ImageJ version 1.52p (National Institutes of Health, Bethesda, MD, USA).