FIGURE 10.

Knockdown of endogenous OS9 increases NKCC2 biogenesis. A, endogenous OS9 interacts with immature NKCC2. HEK cells overexpressing Myc-NKCC2 were immunoprecipitated (IP) with anti-Myc (positive control; lane 1), anti-V5 (negative control; lane 2), or anti-OS9 antibody (lane 3). Co-immunoprecipitated NKCC2 (mainly the immature form) was detected by immunoblotting (IB) using anti-Myc antibody (lane 3). IgGH, the heavy chain of IgG. B and C, knockdown of endogenous OS9 increases total and cell surface expression of NKCC2. Upper panels, representative immunoblotting analysis illustrating the effect of OS9 knockdown on total and surface NKCC2. HEK cells were transfected with NKCC2 in the absence (Control) or presence of specific OS9 siRNAs from Dharmacon (D) or Santa Cruz Biotechnology (S). 48 h post-transfection, biotinylated proteins were recovered from cell extracts by precipitation with NeutrAvidin-agarose. An aliquot of the total cell extract from each sample was also run on a parallel SDS gel and Western blotted for total NKCC2 expression. NKCC2 proteins were detected by immunoblotting with Myc antibody. Lower panels, summary of results. Data are expressed as a percentage of control. *, p < 0.004 versus control (n = 4); #, p < 0.007 versus control (n = 4); §, p < 0.0001 versus control (n = 5); **, p < 0.03 versus control (n = 5). D, knockdown of endogenous OS9 increases NKCC2 stability and maturation. HEK cells were first transfected in the absence (Control) or presence of OS9 siRNA followed by transfection with NKCC2 plasmids 24 h later. 12–14 h post-transfection of NKCC2, cells were chased for the indicated times after addition of cycloheximide. Total cell lysates were subjected to immunoblot analysis for NKCC2 and OS9. Lower left panel, quantitative analysis of immature NKCC2. The density of the immature form of NKCC2 proteins was normalized to the density at time 0 (100%). Lower right panel, quantitative analysis of NKCC2 maturation. The results are presented as relative intensity. Each point represents the mean ± S.E. of three independent experiments. *, p < 0.05 versus controls (n = 4). Error bars represent S.E.