Figure 4. Mutation of PKC putative phosphorylation site (Ser²³⁵) prevents MIP translocation from the trans-Golgi network to the plasma membrane. Immunofluorescence of RK13 cells transfected with pCMV- MIP (WT; A-I) or pCMV-MIP Ala²³⁵ (S235A; J-Q) for 72 h is shown. A and E as well as J and N show the merged images of MIP red immunofluorescence with the corresponding images of their DAPI nuclear staining. B and F as well as K and O show the trans-Golgi network marker 38K (TGN) green immunofluorescence. C, G, L, and P show the merged images of MIP immunofluorescence and DAPI nuclear staining with their respective TGN green immunofluorescence (A and B; E and F; J and K; N and O, respectively). Spatial quantification was performed along a path across the plasma membrane, indicated by a white line with prominent end points in the merged images (C, G, L, and P). Red and green fluorescence was quantified separately and plotted as a function of distance along the path (D, I, M, and Q). Blue broken lines in the spatial quantification graphs indicate the approximate location of the plasma membrane (except the left line in D, which corresponds to the TGN region in C). Note that WT MIP and TGN vesicles colocalize at the plasma membrane (peaks are indicated with blue lines in I and right peak in D). MIP Ala²³⁵ mutant (S235A) does not colocalize with TGN vesicles at the plasma membrane (blue lines; M and Q). C and G show colocalization (yellow) of WT MIP (red immunofluorescence) and TGN 38K (green immunofluorescence) in the cytoplasmic compartment in addition to the localization of WT MIP in the plasma membrane. J and N show MIP Ala²³⁵ mutant punctate distribution in the cytosolic compartment (red immunofluorescence) and colocalization (yellow) with trans-Golgi network 38K (green) in L and P. A and E as well as J and N show cell images from either duplicate experiments or in different fields of the same cell culture of WT or MIP Ala²³⁵ mutant (S235A), respectively. Scale bars represent 10 μm. Note that three cells in A, B, and C (one cell at the right side and two cells in the upper part of the panels) that did not uptake the transfected MIP expression plasmid served as negative controls. They show no red immunofluorescence in contrast to two transfected cells showing the red immunofluorescence. All the cells in the panel (transfected and non-transfected) show green immunofluorescence for TGN38.