(G) Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO (TKO) HeLa cells expressing either wild-type EGFP-GRAM1b (WT) or mutant EGFP-GRAM1b (G187L) as indicated. Cells were cultured in the medium supplemented with 10% lipoprotein deficient serum (LPDS) and mevastatin (50 µM) for 16 hours before imaging. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 36 cells (WT), n = 37 cells (G187L), data are pooled from two independent experiments for each condition; two-tailed unpaired Student's t-test, **P = 0.0003]. Note the rapid PM recruitment of G187L mutant compared to WT. 