Monitoring Rac1 Signaling in U2OS Cells

U2OS cells were transduced with the construct CSCW-Gluc-YFP, as described elsewhere^{44 (link)}. Cells expressing Gaussia luciferase (Gluc) were cultured in 96-well plates at a 10⁵ cells/well density. After 6 h, expression of Flag-RAC1^T17N was induced with 1 μg/ml doxycycline for 22 h. In parallel, cells expressing endogenous RAC1 only were treated with 50 μM NSC 23766 for the last 8 h of culture before measuring luciferase activity. Luciferase activity in the culture medium (secreted Gluc) and the intracellular luciferase activity were evaluated with the Gaussia Luciferase Glow Assay Kit (ThermoFisher Scientific) following the instructions of the manufacturer. Luminescence was measured using a Varioskan Flash luminometer (ThermoFisher Scientific). Controls were done by incubating cells with 5 μg/ml brefeldin A for 2–4 h.