RAB7 and RAB8 are ICMT substrates. (A) ICMT activity of membrane fractions isolated from U2OS cells treated with either NT or ICMT siRNA (i) and SKMEL-28 cells with and without genomic disruption of ICMT (ii) using CRIPSR/Cas9 ([³H methyl-]adenosyl-l-methionine and N-acetyl-S-farnesyl-l-cysteine as a methyl donor and acceptor, respectively). The data shown in i are means ± SEM (n = 3), and those in ii are means of triplicates from a single experiment. Shown below the graphs are representative immunoblots (IBs) of lysates from the same cells using ICMT and β-tubulin antibodies. CPM, counts per minute. (B) Incorporation of l-[methyl-³H]methionine into alkaline-labile [³H]methyl esters on the indicated GFP-tagged proteins expressed in U2OS cells with prior transfection with either NT or ICMT siRNA. Data shown are normalized to the level of protein expression as measured by Western blotting using a GFP antibody and to the amount of incorporation in GFP-NRAS without ICMT knockdown (shown as 100%). Data are representative of two experiments.