LPS induced phosphorylation of MAPKs.

<p>p44/42 ERK (<b>A1–A3</b>) and p38 MAPK (<b>B1–B3</b>) were phosphorylated after CXCL12 (20 nM) stimulation or LPS (2 ng/ml) treatment. Human monocytes (2×10⁶ cells for each time point) treated with or without CXCL12 were collected at 1.5, 2, 3 and 5 min. Monocytes (5×10⁶ for each time point) treated with or without LPS were collected 0, 15, 30, 60 and 100 min. Half of the cells treated with or without LPS and all the cells treated with or without CXCL12 were extracted with RIPA buffer and proteins were resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK (<b>A1</b> and <b>A2</b>). Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (<b>B1</b> and <b>B2</b>). Immunoblots are representative of results with monocytes from 3 donors. The remaining half of the cells treated with or without were stained with a mixture of Alexa-647 E10 mAb against phospho ERK (T202/Y204) and Alexa-488 28B10 mAb against phospho-p38 MAPK (T180/Y182) and analyzed by flow cytometry. Ratios of MFVs for the respective phosho-MAPKs in LPS treated vs. untreated cells are plotted as histograms (n = 5, * p<0.03, ** p<0.01). The remaining cells were extracted with RIPA buffer and proteins resolved by 4–20% gradient SDS-PAGE. Phospho-ERK and total ERKs were detected by immunoblotting with phospho-ERK specific mAb, E10 and rabbit antibody against total ERK. Reacting with anti phospho-p38 MAPK (T180/Y182) mAb and rabbit antibody against total p38 detected phospho-p38 and total p38 respectively (A3 and B3) Immunoblots are representative of results with monocytes from 5 donors.