Loss of ICMT, RAB7, or RAB8 inhibits endogenous NOTCH1 signaling measured as decreased formation of S3 cleaved NICD. (A) Immunoblot for the indicated proteins of MCF10A cell lysates 4 d after treatment with the indicated siRNAs and with or without treatment with media containing 10 mM EDTA for 30 min. Graph to the right shows the normalized ratio of the amount of S3-cleaved NOTCH1 (NICD) to the full-length, uncleaved, S1-processed NOTCH1 (means ± SEM; n = 3). (B) As in A, except using MCF10A with or without genomic disruption of ICMT by CRISPR/Cas9. (C) NOTCH signaling in MCF10A cells quantified with a NOTCH-responsive CSL firefly luciferase reporter. Shown are the ratios of CSL firefly/CMV renilla in MCF10A cells cocultured with 3T3 or 3T3-J2 fibroblasts. The MCF10A cells were transfected with the indicated siRNAs 4 d before the luciferase reading. Values were normalized to the maximum firefly/renilla ratio in NT siRNA–treated cells and are given as means ± SEM. n = 3. *, P < 0.05 (two-sided t test).