Mitogen Activated Protein Kinase Pathway

Extracts prepared from HEK 293 cells treated with UV irradiation were resolved on a 10% polyacrylamide gel and transferred to PVDF. Prior to incubation with p38 MAPK [pTpY180/182] antibody, the immobilized membranes were incubated with the (lane 1) peptide immunogen, (lane 2) a generic phosphothreonine-containing peptide, (lane 3) a generic phosphotyrosine-containing peptide, (lane 4) the non-phosphorylated peptide corresponding to the phosphopeptide, (lane 5) no peptide, (lane 6) the phosphopeptide derived from the corresponding region of JNK1/2, and (lane 7) the phosphopeptide derived from the corresponding region of ERK1/2. The data show that only the phosphopeptide corresponding to p38 MAPK [pTpY180/182] blocks the antibody signal, thereby demonstrating the specificity of the phosphospecific antibody.

FFPE human lung carcinoma tissue was labeled with rabbit anti-ERK1/2 [pT185/pY187] (5 µg/ml). Tissues were pretreated with EDTA, then detected with SuperPicTure™ Polymer DAB . Images were taken at 20x magnification. Note nuclear and cytoplasmic staining in tumor cells.

Recombinant p38 MAPK was phosphorylated in vitro using MKK6 enzyme. Non-phosphorylated p38 MAPK was used as control. The phosphorylated and non-phosphorylated p38 MAPK were analyzed with p38 MAPK [pTpY180/182] ELISA and p38 MAPK (Total) ELISA. Both forms of the p38 MAK protein were detected using the total ELISA whereas only the phosphorylated p38 MAPK protein was detected with the phosphospecific ELISA.