Effect of β2 integrin engagement on the phosphorylation status and activation of ERK1/2 kinase activity.
Panels A to E: Time course of stimulation of the phosphorylation status of ERK1 and ERK2 MAP kinases by β2 integrin engagement. Monocytes (5 × 106 cells) were stimulated for the indicated times with anti-CD11a (5 μg/mL), anti-CD11b (5 μg/mL), anti-CD11c (5 μg/mL), MBP-CD23 (1 μg/mL), and ZZ-CD23 (5 μg/mL), respectively. Cell lysates were analyzed on SDS-PAGE, followed by Western blot using a polyclonal antibody raised against the dually phosphorylated ERK1 (44 kd) and ERK2 (42 kd). Western blots were stripped and reprobed with anti-ERK2 rabbit polyclonal antibody as a loading control. Results are the most representative of 4 distinct experiments. Panel F: Effect of β2 integrin engagement on the activation of ERK1/2 kinase activity. Nonadherent human monocytes (15 × 106 cells) were untreated or stimulated with anti-CD11a (5 μg/mL), anti-CD11b (5 μg/mL), anti-CD11c (5 μg/mL), ZZ-Eselectin (5 μg/mL), ZZ-CD23 (5 μg/mL), or MBP-CD23 (2 μg/mL) in the presence or absence of U0126 (20 μmol/L). After 15 minutes of incubation, cell lysates were prepared and immunoprecipitated with antiphospho-ERK1/2 antibody. The pelleted immunoprecipitates were incubated with Elk1-GST fusion protein as a substrate and phosphorylation of Elk1 was visualized by Western blot using an antibody specific for phosphorylated Elk1.
