RAC1 co-precipitated with ORAI1 upon stimulation with EGF. Panel A: Cells were starved overnight in FBS-free cultured medium and then treated with 50 ng/ml EGF. At the indicated times, endogenous RAC1 activation (GTP-bound RAC1) was analyzed with a pull-down assay to evaluate the amount of RAC1 able to bind PAK1-PBD (see Methods section). Total RAC1 was analyzed as a loading control of the immunoblot. The blot is representative of 3 independent experiments and the quantification of active RAC1 was performed by analyzing the 3 independent experiments. Panel B: Cells were transfected for the expression of ORAI1-GFP, starved overnight with FBS-free medium and treated with 50 ng/ml EGF for 3 min. When required, cells were pre-incubated with 50 μM NSC 23766 for 8 h before the treatment with EGF. Then, ORAI1-GFP was pulled down with GFP beads, and the level of endogenous RAC1 co-precipitated was analyzed by immunoblot. The blot is representative of 3 independent experiments. Panel C: Cells were starved overnight in FBS-free cultured medium, treated with NSC 23766 when required, and then treated with 50 ng/ml EGF for 3 min. Inhibition of RAC1 by NSC 23766 was assessed with a pull-down assay to evaluate active RAC1, as in panel A. The blot is representative of 3 independent experiments. Quantification of RAC1 inhibition by NSC 23766 was performed by analyzing the 3 independent experiments. GTP-bound RAC1 levels were normalized with values obtained in the absence of stimulation (time = 0 min). Full-length blots are presented in Supplementary Fig. S13.
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