Fig. 7
The de-phosphorylation of RA-GEF is necessary for Rap1 activation. a Mouse T cells untreated or treated with okadaic acid (OA) or staurosporine were stimulated with CCL21 and subjected to a pull-down assay. Bound Rap1 (Rap1-GTP) and total Rap1 were detected with anti-Rap1. b (Upper left) The de-phosphorylation of RA-GEF-2 in T cells stimulated with or without CCL21 for 60 s in the presence or absence of OA or staurosporine was analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-RA-GEF-2. (Right) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.006 versus unstimulated cells without OA, *2P < 0.01 versus unstimulated cells without OA, *3P < 0.05 versus unstimulated cells with OA, *4P < 0.001 versus CCL21-stimulated cells without OA. (Lower left) The de-phosphorylation of RA-GEF-2 in BAF cells stimulated with or without CXCL12 in the presence or absence of OA or staurosporine was analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-RA-GEF-2. (Right) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.005 versus unstimulated cells without OA, *2P < 0.04 versus unstimulated cells without okadaic acid, *3P < 0.008 versus unstimulated cells with OA, *4P < 0.004 versus CXCL12-stimulated cells without OA. C (Left)The de-phosphorylation of flag-RA-GEF-1 in BAF cells stimulated with or without CXCL12 in the presence or absence of OA or staurosporine was analyzed by Phos-tag (upper) or conventional (lower) SDS-PAGE followed by immunoblotting with anti-flag. (Right) Quantification of ①, ②, and ③ bands, which is presented as percentage of each band. *1P < 0.02 versus unstimulated cells without OA, *2P < 0.03 versus unstimulated cells without OA, *3P < 0.04 versus CCL21-stimulated cells without OA. The Rf value of 1.0 is defined as the position of the BPB dye. A representative of three independent experiments is shown. Each bar graph represents the means ± SEM