Figure 6. Native Ykt6 is doubly prenylated.

• A
  Immunoblot of cell lysates from WT HAP1 cells and PTAR1 KO HAP1 cells using anti‐Ykt6 antibody. Note the slight difference in the gel mobility of Ykt6 (arrows).
• B
  Prenylation status of rat brain Ykt6. Recombinant unprenyl Ykt6, Cys195‐farnesyl Ykt6, Cys194/195‐diprenyl Ykt6 samples (1 ng each), and rat brain cytosol were electrophoresed on a polyacrylamide gel using deoxycholate (DOC)‐containing buffer and analyzed by immunoblotting with anti‐Ykt6 antibody (DOC‐PAGE; upper panel). The same samples were analyzed by conventional SDS–PAGE and immunoblotting with anti‐Ykt6 antibody (SDS–PAGE; lower panel).
• C
  Prenylation status of Ykt6 in WT and PTAR1 KO cells. Recombinant Ykt6 samples and cell lysates of WT cells, PTAR1 KO cells, and PTAR1 KO cells stably expressing PTAR1 (KO + PTAR1) were separated by DOC‐PAGE (upper) or SDS–PAGE (lower), and analyzed by immunoblotting with anti‐Ykt6 antibody.
• D
  In vitro reconstitution of Ykt6 double prenylation. Dialyzed cytosol prepared from PTAR1 KO HAP1 cells was incubated at 37°C for the indicated times with recombinant GGTase‐III (100 nM) or RabGGTase (100 nM) in the absence or presence of GGPP (10 μM). After incubation, reaction products were separated by DOC‐PAGE (upper) or SDS–PAGE (lower), and analyzed by immunoblotting with anti‐Ykt6 antibody.

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