Fig. 6. B16 cells were co-transfected with myc-PIP5K, GFP-mTuba and mRFP-actin. Cells were cultured on glass coverslips and fixed. (A) GFP-mTuba incorporates into comet tails and concentrates at the vesicles. Box indicates region examined in lower panels. (B) PIP5K expression often generated long-tailed comets with mTuba concentrated at the vesicle. Box indicates region examined in lower panels. (C) Schematic representation of targeted sites for RNAi. `A' targets a region in Tuba between the GEF and BAR domains. `B' targets a region between the two C-terminal SH3 domains. (D) B16 cells transfected with control plasmid, RNAi `A' or RNAi `B' were FACS sorted for equivalent expression (50% intensity and above) at 48 hours and lysed 72 hours post-transfection. Cell lysates were subjected to SDS-PAGE and probed with Tuba antibody (Tuba #5415) and ß-actin antibody (loading control). Both RNAi constructs resulted in 75% knockdown of both Tuba and mTuba. (E) B16 cells were transfected with either pRK5myc-PIP5K alone (PIP5K), pRK5myc-PIP5K and Cherry-mTuba (PIP5K + mTuba), pRK5myc-PIP5K and RNAi `A' (PIP5K + RNAi A), or pRK5myc-PIP5K and RNAi `B' (PIP5K + RNAi B). Cells were cultured on glass coverslips and fixed. The number of comets formed per cell was quantitated. RNAi `B' significantly reduced the number of comets observed per cell relative to cells transfected with PIP5K alone. Error bars represent s.d. (***P<0.005, **P<0.01). Bars: A,B, 5 µm.