Fig. 2. Conventional kinesin heavy chain (KHC) is associated with insulin granules in ß-cells. (A) INS-1 cells were homogenised (Hom.) and the phogrin.EGFP-containing vesicles were immunoadsorbed with a monoclonal anti-EGFP antibody (GFP) or with a monoclonal anti-SREBP antibody (Cont.) (for details, see Materials and Methods). Immunoblots were probed with a polyclonal-pan-kinesin antibody (left panel), a rabbit polyclonal anti-ubiquitous conventional KHC antibody (uKHC) (middle panel) or a monoclonal anti-dynein antibody (right panel). 15 µg protein was loaded from the cell homogenates and 2.5 µg from purified kinesin from pig brain (Kin); the protein content of the immunoadsorbed samples was not determined. Note that the immunoreactive band of 60 kDa detected by the anti-dynein antibody in the immunoadsobed sample is corresponds to IgG. (B) INS-1 cells were co-immunostained with a rabbit polyclonal anti-uKHC (1:200) and a guinea pig polyclonal anti-insulin (1:500) antibody and visualised with an Alexa 488 goat anti-rabbit (1:500) and an Alexa 568 goat anti-guinea pig (1:500) secondary antibody. (a) Alexa 488 fluorescence (488 nm excitation). (b) Alexa 568 fluorescence (568 nm excitation). (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in (a-c). Bars, 5 µm (a-c); 0.5 µm (d-f).