FIGURE 1.

Identification of SCAMP2 as a novel NKCC2-interacting protein. A, SCAMP2 interacts with the proximal region of NKCC2 C terminus in yeast. Yeast two-hybrid analysis was performed using the Matchmaker system as described under “Experimental Procedures.” The experiment demonstrates that co-expression of SCAMP2 and C1-term is required to support the growth of yeast in the absence of tryptophan, leucine, histidine, and adenine in the medium. This experiment also demonstrates the specific interaction between C1-term (but not C2-term) and SCAMP2. B, full-length NKCC2 interacts with SCAMP2 in OKP cells. OKP cells transiently expressing NKCC2 and SCAMP2 or NKCC2 alone were immunoprecipitated (IP) with anti-Myc (positive control; lane 4) antibody or anti-V5 anti-body (lanes 5 and 6). NKCC-2 co-immunoprecipitated with SCAMP2-V5 was detected by immunoblotting (WB) using anti-Myc (lane 6). 5% of total cell lysate (Lys) was resolved as positive control. C, endogenous SCAMP2 interacts with NKCC2. Cell lysates from OKP cells transiently transfected with Myc-NKCC2 were immunoprecipitated with anti-Myc or anti-V5 or non-related antibodies (negative control; lanes 2 and 4) or anti-SCAMP2 antibody (lane 3). Co-immunoprecipitated NKCC2 was detected by immunoblotting (lane 3). IgGH, the heavy chain of IgG.