(E) Wild-type (control) and GRAMD1 TKO (TKO) HeLa cells that stably expressed either EGFP or EGFP-tagged GRAMD1b constructs as indicated [wild-type (WT), R189W mutant (R189W), R189W/R191A mutant (RW/RA)], were cultured in the medium supplemented with 10% lipoprotein-deficient serum (LPDS) and mevastatin (50 µM) for 16 hours and then treated with SMase (100 mU/ml) for 3 hours at 37°C. Top: Lysates of the cells were processed for SDS-PAGE and IB with anti-SREBP-2 and anti-actin antibodies. Arrows indicate precursor (P) and cleaved (C) forms of SREBP-2. Bottom: The response rate was obtained by normalizing the ratio of the band intensity of the cleaved SREBP-2 over the total band intensity of cleaved and precursor forms of SREBP-2 from the cells with SMase treatment by the one from the cells without SMase treatment for each condition. Note that the suppression of SREBP-2 cleavage is attenuated in GRAMD1 TKO HeLa cells compared to wild-type control HeLa cells. Note also the rescue by expression of wild-type EGFP-GRAMD1b (WT) but not by mutant versions of EGFP-GRAMD1b [R189W, R189W/R191A (RW/RA)] [mean ± SEM, n = 4 lysates (independent experiments) for each condition; Dunnett's multiple comparisons test, **P = 0.0035 (Control + EGFP vs. TKO + EGFP), **P < 0.0001 (TKO + EGFP vs. TKO + WT), n.s. denotes not significant]. 