(B) Confocal images of live GRAMD1 TKO HeLa cells stably expressing EGFP-GRAMD1b constructs as indicated that were additionally transfected with an accessible PM cholesterol biosensor, mCherry-tagged GRAM domain of GRAMD1b (mCherry-GRAM1b). Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Note the very weak PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b WT, compared to the strong PM recruitment of mCherry-GRAM1b in cells that stably expressed EGFP-GRAMD1b R189W mutant (R189W) or EGFP-GRAMD1b R189W/R191A mutant (RW/RA). Scale bars, 10 µm. 