Figure 2.

PI4Kβ/rab11 association is independent of lipid kinase activity. (A) For yeast two-hybrid assay, S. cerevisiae AH109 cells were transformed with the kinase dead mutant PI4KβD656A in the bait vector pGBT9 (pGBT9-PI4KβkDa), and rab11A or rab11B in pACT2, or with pACT2 alone as control (empty). Yeast cells were grown for 4 d on selective medium (-Trp, -Leu, -Ade), and images of culture plates are shown. (B) COS-1 cells were transfected with 5 μg of cDNA encoding Myc-tagged proteins PI4Kβ or PI4KβkDa, and detergent lysates (1 ml) were used in GST pull-down assays with 5 μg of GST or GST-rab11. Proteins bound to GSH beads were split, either analyzed by Western blotting by using anti-Myc (WB, pull down), or subjected to an in vitro lipid kinase assay (TLC). As controls, PI4Kβ and PI4KβkDa present in cell lysates were detected with anti-Myc (WB, lysate), and GST-fusion proteins were stained with Coomassie Brilliant Blue (CBB).