FIGURE 5.

SCAMP2 co-localizes with NKCC2 in Rab11-positive compartments. A, endogenous SCAMP2 co-localizes with NKCC2. HEK cells transiently transfected with Myc-NKCC2 were double-stained with mouse anti-Myc (Texas Red) and rabbit anti-SCAMP2 (FITC) antibodies. Yellow, overlap between the Myc tag of NKCC2 protein (red) and SCAMP2 (green), representing co-localization of the proteins. B and B′, immunofluorescence confocal microscopy showing distribution of NKCC2 and SCAMP2 in HEK and OKP cells. OKP cells were transfected with NKCC2 N-terminally tagged with EGFP (green) or Myc with SCAMP2-V5. Fixed and permeabilized cells were stained with rabbit anti-Myc for NKCC2 (green) or mouse anti-V5 for SCAMP2 (Texas Red). The yellow color (merged image) indicates co-localization of the proteins. C, C′, D, D′, E, E′, F, G, and H, comparison between the distribution of NKCC2 and SCAMP2 and that of the recycling endosomal marker Rab11 and internalized transferrin. Transiently transfected cells with Myc-NKCC2 and/or SCAMP2-V5 proteins were stained with mouse anti-Myc for NKCC2 (Texas Red) or mouse anti-V5 for SCAMP2 (Texas Red) and rabbit anti-Rab11 (FITC, green). Internalized biotinylated holotransferrin was revealed with avidin-Cy2 (green). I, comparison between the distribution of NKCC2 and SCAMP2 and that of Rab4 (FITC, green). Yellow (merged image), co-localization of the proteins.