The effect of STCH on NKCC2 expression is independent of the expression system. (A) STCH interacts with immature NKCC2 in OKP cells. Cells were transiently transfected with Myc-NKCC2 either singly or in combination with GFP-STCH construct. Cell lysates were immunoprecipitated (IP) with anti-GFP antibody. NKCC2 protein was recovered from STCH immunoprecipitates mainly in its immature form (lane 3). (B) Imunofluorescence confocal microscopy showing distribution of Myc-NKCC2 and GFP-STCH in OKP cells. Cells were stained with mouse anti-Myc for NKCC2 (Texas Red). The yellow color (merged image) indicates co-localization of the proteins. Bars, 5 μm. (C) Similar to HEK cells, STCH and NKCC2 co-localizes mainly at the ER in OKP cells. All panels are fluorescence micrographs of OKP cells overexpressing myc-NKCC2 and GFP-STCH. Upper panel, fixed and permeabilized cells were stained with mouse anti-Myc and rabbit anti-calnexin (ER marker) antibodies. The merge color indicates overlap between the Myc tag of NKCC2 protein (Alexa Fluor 647, Blue), the GFP tag of STCH (green), and the ER marker (Alexa Fluor 555, red) and represents co-localization of the proteins. In lower panel, fixed and permeabilized cells were stained with mouse anti-Myc and rabbit anti-GM130 (cis-Golgi marker) antibodies. The merge color indicates overlap between the Myc tag of NKCC2 protein (Alexa Fluor 647, Blue), the GFP tag of STCH (green), and the cis-Golgi marker (Alexa Fluor 555, red) and represents co-localization of the proteins. In addition to the ER, the interaction between NKCC2 and STCH may also occur at the cis-Golgi. Analysis was performed by confocal laser scanning microscopy. Bars, 5 μm. (D) Analysis of NKCC2 stability and maturation monitored by cycloheximide-chase upon STCH expression. OKP cells were co-transfected with NKCC2 together with a control vector or GFP-STCH construct. Then, 14 h later, cell lysates were prepared at the indicated time points after cycloheximide treatment (100 μM). Total protein extracts are subjected to SDS-PAGE and probed using anti-Myc antibody. The density of the mature and immature forms of NKCC2 proteins was normalized to the density at time 0. #, *; p < 0.05 (n = 3) versus control.