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. Author manuscript; available in PMC: 2020 Mar 15.
Published in final edited form as: J Immunol. 2019 Feb 1;202(6):1815–1825. doi: 10.4049/jimmunol.1801477

FIGURE 5. MEK2 is dispensable for LPS mediated MAP kinase activation including ERK phosphorylation.

FIGURE 5.

Murine BMDMs derived from WT, and Mek2−/− mice were treated with LPS (100 ng/mL) for different time points as indicated. (A) Detection of MEK1 and MEK2 isoforms. Whole cell extracts were prepared and subjected to SDS-gel electrophoresis and Western blot analysis using antibodies against MEK1 and MEK2. Equal loading was confirmed using β-actin antibodies. As shown, MEK2−/− BMDMs lack expression of MEK2. (B) LPS induced phosphorylation of MEK1/2 in WT and MEK2−/− BMDMs. Murine BMDMs were challenged with LPS for different time points as indicated. Whole cell extracts were prepared and 15μg of proteins were subjected to SDS-gel electrophoresis and Western blot analysis was performed using antibodies against the phosphorylated forms of MEK1/2 (Ser217/221). Equal loading was confirmed using β-actin antibodies. MEK2−/− BMDMs showed a lower phosphorylation of MEK1/2 which is mainly due to phosphorylation of MEK1. (C) LPS induced phosphorylation of ERK. Whole cell extracts were subjected to SDS-gel electrophoresis and Western blot analysis performed using antibodies against the phosphorylated forms of ERK (Thr202/Tyr204) and total ERK. (D) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total ERK. (E) LPS induced phosphorylation of JNK. Western blot analysis was performed with antibodies against the phosphorylated form of SAPK/JNK (Thr183/Tyr185) and equal loading was determined measuring total JNK. (F) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total JNK. (G) LPS induced phosphorylation of p38. Western blot analysis was performed with antibody against the phosphorylated form of p38 (Thr180/Tyr182) and equal loading was determined using antibody against total p38. (H) Densitometric analysis of at least 3 independent experiments expressed as fold change of the ratio phosphorylated/total p38. Using ANOVA Mann-Whitney U test, a p value <0.05 was considered significant and error bars indicate SEM.