Inp54p translocates to plasma membrane in HEK293 cells, reducing PIP
biosensor levels following rapamycin treatment. A-F) HEK293 cells were transfected with plasmids encoding FRBPLF-CFP, Venus-FKBP12-Inp54p and PLC∂1PH-RFP, a PIP2 biosensor. One day later, cells were imaged by live-cell confocal microscopy. Before rapamycin treatment, (A) FRBPLF-CFP, (B) Venus-FKBP12-Inp54p, and (C) PLC∂1PH-RFP were properly localized to the (A,C) membrane or (B) cytoplasm. While localization of FRBPLF-CFP remains constant, rapamycin (1 μM) induced rapid (<3 s) translocation of (E) Venus-FKBP12-Inp54p to the plasma membrane and (F) reduced PLC∂1PH-RFP levels at the membrane, indicative of PIP2 hydrolysis. G) Quantification of translocation in HEK293 cells. **P < 0.005 compared to pre-rapamycin condition, n = 20 cells per condition. Scale bar, 10 μm.