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Figure 2


Fig. 2. Subcellular distribution of SNARE and SNARE-regulator Munc18 proteins in MCD4 cells. Subcellular localization of SNARE and Munc18 isoforms was analyzed in MCD4 cells by CLSM and western blotting. (A-G) Polarized MCD4 cells were fixed and stained with antibodies against VAMP2, VAMP3, Stx3, Stx4, SNAP23, Munc18b and Munc18c (all in green) and counterstained with WGA-TRITC (red). Confocal scans were taken in the yz plane to analyze the apical vs basolateral distribution of each protein. (A'-G') MCD4 total homogenates, plasma membrane (P.M.), intracellular vesicles (I.V.) and mouse kidney homogenates were separated by NuPAGE electrophoresis and analyzed by western blotting using the same antibodies as above. VAMP2 and VAMP3 were found almost equally distributed between plasma membranes and intracellular vesicles, whereas Stx3, Stx4 localized at the apical and basolateral membrane respectively. SNAP23 was abundantly expressed at both the plasma membrane and intracellularly. Munc18b and Munc18c were both associated with the apical plasma membrane. Same results were obtained in at least three independent experiments.





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