Transfection with constitutively active PLC induces ERM protein dephosphorylation and release from the plasma membrane. (A) Jurkat cells were transfected with the indicated PLC-γ1 constructs (NN, constitutively active; or Y783F, inactive) or construct-encoding GFP-PH and analyzed by WB. Expression of PLC-γ1 in Jurkat does not influence the expression of moesin constructs (Fig. S3, available). (B) Imaging of control cells (top) compared with cells transfected with constitutively active NN PLC-γ1 construct (middle) or with inactive PLC-γ1 Y783F (bottom). Markers examined are the transfected PLC (anti-HA antibody, purple), pERM level (red), and transfected GFP-PH localization. (C) Fluorescent analysis of Jurkat cells cotransfected with GFP-moesin (wt or T558D) and PLC-γ1 constructs (detected with anti-HA antibody [red]). (D) Quantitative analysis of data from C (n = 10 for each condition). Quantitative analysis was performed as described in Materials and methods. (E) SDF-1 stimulation induces hydrolysis of PIP2. Jurkat cells were transfected with GFP-tagged PH domain construct and analyzed by immunofluorescence microscopy after SDF-1 or PLC activator treatment. Note that unlike PBTs, Jurkat cells do not undergo striking shape change (polarization) in response to SDF-1. Error bars indicate SEM. DIC, differential interference contrast.