CRP induces PI 3-kinase–dependent translocation of PLCγ2 and Btk to plasma membrane.

(A) Micrographs show the localization of PLCγ2 and Btk in wild-type Balb-c mouse megakaryocytes. The megakaryocytes have been preincubated with wortmannin (wort) (100 nM) or LY294002 (50 μM) for 15 minutes and then activated with CRP (4 μg/mL) for 150 seconds. Cells were fixed and stained for PLCγ2 and Btk as described in “Materials and methods.” Distribution was analyzed by fluorescence microscopy and deconvolution for basal, CRP-activated megakaryocytes; wort-pretreated megakaryocytes; and LY294002 (LY)–pretreated megakaryocytes. Scale bar = 20 μm. (B) Distribution of fluorescence intensity in megakaryocytes along the lines shown in panel A was calculated by densitometry. The distributions in (i) and (ii) correspond to basal- and CRP-stimulated samples, respectively. (C) The time course of protein translocation was measured in megakaryocyte in response to CRP (4 μg/mL). The results are shown as distribution ratio (Ipm/Icyt) over time (described in “Results”). (D) Control and wort- or LY294002-pretreated megakaryocytes were stimulated by CRP for 150 seconds, and cells stained for PLCγ2 or Btk as described in “Materials and methods.” The amount of protein at the membrane was calculated as above. All results are expressed as a mean ± SE, and are from at least 8 megakaryocytes from 3 different experiments. * and ** correspond to results significantly different from basal or after stimulation with CRP for 2.5 minutes, respectively.