Fig. 3. Generation and expression of KHC^mut conventional kinesin heavy chain construct. (A) cDNA encoding the rat conventional kinesin heavy chain motor domain containing a T93N point mutation at the ATP-binding consensus motif, and a histidine-tag (6His) was cloned into the XbaI-XhoI sites of pcDNA3.1(-). INS-1 cells were transfected, fixed 48 hours later with cold methanol and then co-immunostained with a rabbit polyclonal anti-6His-tag antibody (1:250) (a) and a mouse monoclonal anti -tubulin antibody (1:1000) (b) and visualised with Alexa 568 and -488 secondary antibodies, respectively. (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in (a-c). Bars, 2.5 µm (a-c); 1.1 µm (d-f). (B) Cells were co-transfected with KHC^mut-pAdTrack-CMV and mitochondrial.DsRed. 48 hours after transfection cells were imaged on a confocal microscope. The KHC^mut-expressing cells were identified by exciting EGFP at 488 nm and the DsRed fluorescence of the same cells was visualised by exciting at 568 nm. Typical DsRed in vivo confocal images of mitochondria in INS-1 (a,b) and in HeLa cells (c,d) in KHC^mut expressing (a,c) and control cells (b,d) are shown. Scattered lines indicate the position of the plasma membrane obtained as an overlay from the transmitted image of the cell. Bars, 2.5 µm (a-d).