FIGURE 1.

Identification of OS9 as a novel NKCC2-interacting protein. A, OS9 interacts specifically with the distal region of NKCC2 C terminus in yeast. A yeast two-hybrid assay was performed using the Matchmaker system as described under “Experimental Procedures.” The experiment confirmed the interaction of OS9 with the distal region of NKCC2 C terminus 5 (C3-term) as judged by growth of the AH109 reporter strain on selection medium (−Trp, −Leu, −His, −Ade). In contrast, no growth was observed when AH109 was transformed with OS9 and C1-term. B, NKCC2 interacts in vivo with OS9 in OKP cells. OKP cells transiently expressing Myc-NKCC2 singly or in combination with OS9 were immunoprecipitated (IP) with anti-V5 anti-body (lanes 2 and 3). 5% of total cell lysate (Lys) was resolved as a positive control. NKCC-2 co-immunoprecipitated with OS9 was detected by immunoblotting (IB) using anti-Myc (lane 3). The positions of the core glycosylated (immature) and complex glycosylated (mature) proteins of NKCC2 are indicated. The interaction of NKCC2 with OS9 involves mainly the core glycosylated, immature form of the co-transporter. SD, synthetic defined medium; IgGH, the heavy chain of IgG.