Figure 7. Inhibition of p38 MAP kinase effectively blocks LPS mediated pro-IL-1β, and HIF-1α protein levels as well as ROS production in WT and MKP-1 deficient BMDMs.

(A) WT and MKP-1 deficient BMDMs were cultured in the presence or absence of SB203580 (10 μM, 30 min.) followed by a challenge with LPS (100 ng/mL) for 30 min. Whole cell extracts were prepared and 15 μg total proteins were subjected to SDS-PAGE and Western blot analysis using specific antibodies against phospho p38, equal loading was confirmed using an antibody against total p38. Pretreatment with SB203580 significantly decreased p38 phosphorylation in response to LPS both in WT and MKP-1^−/− BMDMs. (B) WT and MKP-1 deficient BMDMs were cultured in the presence or absence of SB203850 (10 μM, 30 min.) followed by a challenge with LPS (100 ng/mL) for 3h. Whole cell extracts were prepared and 15 μg total proteins were subjected to SDS-PAGE and Western blot analysis using specific antibodies against pro-IL-1β and HIF-1α. Equal loading was confirmed with β-actin. Pretreatment with SB203850 significantly decreased both LPS-induced pro-IL-1β and HIF-1α expression (C) Densitometric values expressed as fold increase of the ratio of pro-IL-1β/β-actin. (D) Densitometric values expressed as fold increase of the ratio of HIF-1α/β-actin. (E&F) BMDMs derived from MKP-1^−/− mice were pretreated with SB203850 inhibitor (10 μM) for 30 min prior to LPS (100 ng/mL) challenge for 1 h followed by the measurement of ROS production using (E) H₂DCFDA and (F) MitoSOX fluorescence intensity. Results show that the cytosolic and mitochondrial ROS production induced by LPS was inhibited by SB203850 pretreatment in MKP-1^−/− BMDMs (n=3).