Fig. 5
Subcellular distribution of NHE6 ΔES is altered in AP-1 cells. a Plasma membrane location of NHE6v1 as measured biochemically using a cell-surface biotinylation assay. To detect cell surface expression of NHE6v1, a triple Flag epitope-tag was inserted into the first predicted extracellular loop of NHE6v1HA (3FNHE6v1HA), as illustrated in the upper panel. AP-1 cells were transiently transfected with 3FNHE6v1HA WT or ΔES for 36 h and cell surface proteins were labeled with biotin as described in ‘Material and Methods’. Total cell lysates (TCL) were prepared and a small portion representing the total fraction was removed. The remaining supernatants containing equal amounts of total protein for WT and ΔES were loaded onto NeutrAvidin® Agarose beads to purify the biotinylated cell surface proteins from the non-biotinylated (intracellular) proteins. For the TCL and the remaining non-biotinylated fractions, aliquots containing 20 μg and 80 μg protein for WT and ΔES, respectively, were examined by Western blotting (left and right lower panels, respectively). For the plasma membrane fraction, 25 % and 100 % of the biotinylated proteins extracted from the total cell lysates of WT and ΔES transfectants, respectively, were subjected to Western blotting (middle lower panel). All immunoblots were probed with mouse monoclonal anti-HAm antibody to detect NHE6 and anti-GAPDHm antibody to assess the enrichment of the biotinylated fraction, as GAPDH is a cytosolic protein. b Confocal fluorescence microscopy and transmitted light images of fixed non-permeabilized AP-1 cells showing surface expression of 3FNHE6v1HA WT or ΔES. Scale bars represent 5 μM. c AP-1 cells were transiently transfected with 3FNHE6v1HA WT (upper panels) or ΔES (lower panels). Thirty-six h after transfection, cells were loaded with Alexa Fluor594-labelled transferrin (Tf-AF594, 10 μg/mL) for 45 min, fixed in 4 % paraformaldehyde, permeabilized, mounted onto glass slides and then examined by confocal microscopy. Footprints of the transfected cells are indicated as white dotted outlines and were derived from the transmitted light images (far left panels). Scale bars represent 10 μm