FIGURE 9.

The effect of OS9 on NKCC2 ERAD is independent of the expression system. A, OS9 interacts with immature NKCC2 in HEK cells. Cell lysates from HEK cells transiently transfected with Myc-NKCC2 singly or in combination with OS9-V5 were immunoprecipitated (IP) with anti-Myc or anti-V5 antibody. NKCC2 protein was recovered from OS9 immunoprecipitates mainly in its immature form (lane 3). B, OS9 and NKCC2 co-localizes in the ER. Upper panel, immunofluorescence confocal microscopy showing the distribution of NKCC2 and OS9 in HEK cells. HEK cells were transfected with NKCC2 N-terminally tagged with EGFP (green) and OS9-V5. Fixed and permeabilized cells were stained with mouse anti-V5 for OS9 (Texas Red). The yellow color (merged image) indicates co-localization of the proteins. Lower panel, HEK cells transfected with NKCC2 and OS9-V5 were stained with mouse anti-V5 (Texas Red; red) and rabbit anti-calnexin (FITC; green). Yellow indicates overlap between the V5 tag of OS9 (red) and the ER marker (green), representing co-localization of the proteins. C, OS9 decreases the steady-state protein level of NKCC2. Cells were transfected with Myc-NKCC2 alone or in the presence of OS9-V5. 48 h post-transfection, total cell lysates were subjected to immunoblotting analysis for Myc-NKCC2, OS9-V5, and actin. D, summary of results. Data are expressed as a percentage of control ±S.E. *, p < 0.02 versus control (n = 3). Error bars represent S.E.