ROD binds the Spindly farnesyl group. (A) Chemical structure of Fpp analogues incorporating photoactivatable cross-linking groups. (B) Schematic of the UV-induced RZZ–Spindly^Farn cross-linking experiment. PP, pyrophosphate. (C) FTase was incubated with Spindly (also in presence of RZZ) to incorporate farnesyl or a modified farnesyl harboring the cross-linking group BPP1 in Spindly. After separation by SEC, elution fractions were subjected to UV irradiation and visualized by Coomassie staining of SDS-PAGE gels. UV-induced cross-linked bands (the most prominent of which are indicated by red arrows) appear in the sample containing BPP1. Equivalent experiments with BPP2 and diazirine are shown in Fig. S4 (A–H). (D) Western blotting (WB) analysis of the indicated selected fractions (from SDS-PAGE in C) with antibodies against ROD, ZW10, Zwilch, and Spindly. Molecular mass is indicated in kilodaltons. (E and F) MS identifies UV-induced cross-links of Spindly’s farnesyl diazirine with residues Leu120 of ROD (shown in red). For each panel, left traces show the chromatographic peak and right traces show the isotopic pattern of the corresponding peptide identified in the MS survey scan. Upper traces represent samples before UV treatment (−UV) and lower traces after UV treatment (+UV). Corresponding MS/MS spectra containing sequence information are shown in Fig. S3 E. Equivalent MS analyses with BPP1 and BPP2 are shown in Fig. S4 (I–L) and MS/MS spectra in Fig. S3 (C and D). RA, relative abundance.