FIGURE 8.

SCAMP2 does not affect NKCC2 internalization. A, typical blot. 48 h post-transfection of OKP cells with Myc-NKCC2 alone (with pcDNA empty vector) or with SCAMP2-V5, cell surface biotinylation was performed with sulfo-NHS-SS-biotin, followed by chase incubation in the culture media at 37 °C for 15 and/or 30 min to permit endocytosis of labeled proteins. Following the chase period, surface biotin was removed by incubation with MesNa, allowing visualization of internalized protein. Cells were then lysed; biotinylated proteins were purified by incubation with avidin-conjugated agarose beads and resolved by SDS-PAGE; and surface-labeled and -internalized Myc-NKCC2 was detected by Western blotting using an anti-Myc antibody (surface NKCC2). A small amount of total lysate (7%) was analyzed as a loading control and probed for SCAMP2-V5 and Myc-NKCC2 (total NKCC2, SCAMP2-V5). B, summary of all experiments (n = 3 for each group). The amount of Myc-NKCC2 internalized at each time point in pcDNA3- or SCAMP2-V5-transfected cells was compared directly with the untreated sample (basal surface NKCC2) from the same experiment, and therefore values are expressed as the mean after normalizing to basal levels. NS, not significant. Error bars, S.E.