Fig. 3
From: C3G, through its GEF activity, induces megakaryocytic differentiation and proplatelet formation
PMA induces C3G phosphorylation in K562 cells, which correlates with a sustained Rap1 activation. a Left panel: time course Western blot analysis of phospho-Y504-C3G (p-C3G) expression in non-transfected K562 cells treated with 20 nM PMA for 2, 5, 10, 30 and 60 min. The expression of total C3G and tubulin were used as loading controls. The asterisk indicates a non-specific band. Right panel: Histogram showing pY504-C3G/C3G ratios, relativized to control, non-treated cells. b Representative immunofluorescence confocal microscopy images of the indicated K562 clones treated with 20 nM PMA for 2, 5 and 10 min, fixed, permeabilized and incubated with: anti-phospho-C3G/anti-rabbit Cy3 antibodies (green), purified GST-RalGDS-RBD and anti-GST/anti-mouse Cy5 antibodies (red) to detect active Rap1-GTP, and DAPI (blue). The overlay images are made without DAPI channel. Images of GFP expression are shown. Histograms represent the mean ± SEM of the integrated density (I.D.) of p-C3G (left panel) and Rap1-GTP staining (right panel). The ANOVA analysis indicates that there is a significant variability between pLTR2-C3G and pLTR2-CT in the fluorescence intensities of p-C3G and Rap1-GTP, independently of the treatment used (p < 0.05). In addition, Holm-Sidak method was done. *p < 0.05