Fig. 5. Rescue of TGN-associated clathrin by mRFP-GAK in GAK-depleted cells. Both mock-depleted and GAK-depleted cells were transfected with mRFP-GAK. After immunostaining with clathrin antibodies, the cells were imaged using the confocal microscope to determine whether there was pronounced perinuclear clathrin staining associated with the TGN. Cells were GAK depleted using the 3'-UTR construct. (A) Mock-depleted (a,b) and GAK-depleted (c,d) cells were imaged for both mRFP-GAK (a,c) and clathrin (b,d). (B) Cells from two experiments (a minimum of 50 cells per experiment), were counted after being imaged using either clathrin immunostaining or GFP-clathrin to determine whether there was pronounced TGN-associated clathrin.