Figure 1.

PKD isoforms interact with ARF1. (A) Lysates of HEK293-T-cells expressing GFP-PKD1 or EGFP-PKD2 or GFP-PKD3 were incubated with GST-ARF1 immobilized on glutathione-Sepharose beads, and retained PKD1, -2, or -3 was assessed by Western blotting with GFP antibody. (B) HeLa cell lysate was incubated with GST-ARF1 immobilized on glutathione-Sepharose beads, and retained endogenous PKD2 was assessed by Western blotting with PKD2 antibody. (C) Exogenously expressed PKD2 and ARF1 interact. Myc (lane 2) or Myc-ARF1 (lanes 1 and 3) was coexpressed with EGFP (lane 1) or EGFP-PKD2 (lanes 2 and 3) in HEK293-T-cells. The cells were immunoprecipitated with anti-Myc antibody (top left) or anti-GFP antibody (top right) followed by anti-GFP (top left) or anti-Myc (bottom right) Western blotting, respectively. To verify that each tagged protein was expressed and immunoprecipitated, the Myc and the GFP precipitates were blotted with anti-Myc (bottom left) and anti-GFP, respectively (top right). (D) Lysates of HEK293-T-cells expressing EGFP-PKD2 were incubated with GST-ARF1-WT or mutants immobilized on glutathione-Sepharose beads. Retained PKD2 was assessed by anti-GFP Western blotting. Quantification of the band intensities of PKD2 bound to GST-ARF1-WT or mutants is represented in the bottom panel. Data are means ± SEM of two independent experiments.