P44 42 mapk rabbit mab

The following antibodies were used for western blot analysis: Rabbit anti-rat ANX A1 (ab65844) was obtained from Abcam (Cambridge, USA); and phospho-p44/42 MAPK (Thr202/Tyr204) rabbit mAb (catalogue no. 4370); p44/42 MAPK rabbit mAb (catalogue no. 4695); phospho-p38 MAPK (Thr180/Tyr182) rabbit mAb (catalogue no. 4511); p38 MAPK rabbit mAb (catalogue no. 8690); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) rabbit mAb (catalogue no. 5174) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). All the primary antibodies were used at a dilution of 1:1,000. The secondary anti-rabbit IRDye 800CW antibody was obtained from LI-COR Biosciences (Lincoln, NE, USA).

Cells were lysed using tissue protein extraction reagent (Thermo, USA) containing phosphatase inhibitor cocktail (1:100, Thermo), protease inhibitor cocktail (1:100, Thermo), and 5 mM EDTA (Thermo); lysates were oscillated and centrifuged (13,000×g, 15 min, 4 °C). The supernatant was collected and stored at − 80 °C. The protein concentrations were determined using the BCA protein assay kit (Thermo). The protein was mixed with 5X SDS-PAGE protein loading buffer (Beyotime, China) and denatured by 100 °C water bath. Then, the samples denatured were electrophoresed in 10% SDS-PAGE and transferred to PVDF membranes using transfer device (Bio-Rad). The membranes were blocked with 5% non-fat milk prepared in TBST for 1 h at 37 °C and then incubated at 4 °C overnight with the primary antibodies: ERK1/2 (1:1000, Cell Signaling Technology, p44/42 MAPK Rabbit mAb, 4695), p-ERK1/2 (1:1000, Cell Signaling Technology, Phospho-p44/42 MAPK (ERK1/2) XP®, Rabbit mAb, 4370), and the internal normalization mouse anti-GAPDH (1:1000, Santa Cruz Biotechnology). Next, the membranes were washed in TBST, incubated with secondary antibody: Goat anti-Rabbit IgG (H + L) IRDye 800CW or Goat anti-Rabbit IgG (H + L) IRDye 800CW (1:20,000; LI-COR) for 1 h at 37 °C. The images were observed with a UVA Bio Imaging System and analyzed with ImageJ software.