(C) Quantification of the ratio of PM mCherry-GRAM1b signals to the cytosolic mCherry-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing mCherry-GRAM1b with SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (B) and Fig EV3B (mean ± SEM, n = 20 cells for each condition; data are pooled from two independent experiments; Dunnett's multiple comparisons test, **P < 0.0001. n.s. denotes not significant). 