Figure 3.

Comparison of metabolic incorporation analysis of C10AlkOPP, C15AlkOPP, C15hAlkOPP, C20AlkOPP probes and pro-analogue counterparts. (A) The metabolic labeling workflow for treating cells with analogous with or without lovastatin. The treated cells are lysed then undergo a copper click reaction with TAMRA-Azide (37). The fluorescently tagged proteins are then visualized in-gel to examine success of metabolic incorporation. (B) In-gel fluorescence analysis of metabolic incorporation of 10 μM C10AlkOPP, C15AlkOPP, C15hAlkOPP, or C20AlkOPP with (odd lanes) or without (even lanes) a 6 h Lovastatin (10 μM, 36) into COS7 cells. (C) COS7 treated in the same manner but with 10 μM of C10AlkOH, C15AlkOH, C15hAlkOH, and C20AlkOH instead, with (even lanes) or without (odd lanes) lovastatin pretreatment. In both the top panel for both is a measurement of TAMRA fluorescence, bottom panel is total protein loading visualized with Coomassie stain. Two regions of note are highlighted with brackets: Blue is the 20–25 kDa region which is where many small GTPases and Rab proteins migrate, common GGTase I and II substrates, and Red which is the 40–75 kDa region where a few more distinctive higher molecular weight FTase substrates appear.