Fig. 8. Expression and effect of KHC340 on vesicle movements and secretion. (A) KHC340 construct was generated as KHC^mut described under Fig. 3. (B) INS-1 cells were transfected with the KHC340-pcDNA3.1(-), fixed 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. Bar, 2.5 µm (a-c). (C) Cells were co-transfected with KHC340-pAdTrack-CMV and phogrin.DsRed. Typical 568 nm in vivo confocal images of insulin-containing vesicles (a,c) in KHC340-expressing (a) and control cells (c). Movements of vesicles were imaged and analysed as described in Fig. 5. (b,d) Tracks of granules in a and c. Bars, 2.5 µm (a,c); 1.95 µm (b,d). (D) Effect of KHC340 on glucose-stimulated human growth hormone (hGH) release from MIN6 cells was studied as described in Fig. 7.

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