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. Author manuscript; available in PMC: 2016 Apr 2.
Published in final edited form as: Mol Cell. 2015 Mar 12;58(1):110–122. doi: 10.1016/j.molcel.2015.01.040

Figure 6. IpaJ cleaves only Golgi-localized GTPases.

Figure 6

(A) Glutathione-pulldown of GST-ARF6Δ13 in the nucleotide free (free), GTP, or GDP bound state in presence of MBP-IpaJΔ50 (left) and visualized as in Figure 5A.

(B) In-gel fluorescence showing myristoylated ARF1 or ARF6 after co-expression with IpaJ or IpaJC64A in HeLa cells (top panel). Equal loading of ARF1/ARF6 (middle panel) and expression of IpaJ (lower panel) were confirmed by western blot analysis. Graph shows the quantification of fluorescent signal (in arbitrary units (au) +/− SD) from three experimental repetitions using ImageJ.

(C) Fluorescent Microscopy of EGFP-tagged ARF1-, ARF5-, ARL1-, and ARF6 when co expressed with mCherry vector (control) or IpaJ-mCherry (IpaJ). Inset box shows ARF protein localization (middle panel) and cis-Golgi (right panel) visualized by gm130 immunofluorescence. The bottom two rows focus on the plasma membrane localization of ARF6.

(D) Fluorescent Microscopy mCherry tagged IpaJC64A and EGFP-tagged ARF1 in HeLa cells left untreated (top row) or treated with Brefeldin A (bottom row). Cis-Golgi was visualized with gm130 antibodies.

(E) Fluorescent Microscopy mCherry tagged IpaJC64A and EGFP-tagged ARF6 in HeLa cells. Inset (1) is focused on the cis-Golgi and inset (2) is focused on plasma membrane.