Figure 4

Analysis of interactions between MCEPH and Kv1 subunits. A. Immunoprecipitation was performed using a monoclonal Kv1.1 C-terminal antibody on brain lysate from wild type (+/+) and Kv1.1 null (-/-) mice. In wild type brain both Kv1.1 and Kv1.2 are detected. In Kv1.1 null brain neither Kv1.1 nor Kv1.2 was detected. B. Immunoprecipitation was performed with the anti-Kv1.2 monoclonal antibody on brain lysate from wild type (+/+) and mceph/mceph (m/m) mice. The immunoprecipitation reaction and corresponding brain lysate was loaded on SDS-PAGE. In brain lysate from mceph/mceph (BL) MCEPH was detected using the polyclonal Kv1.1 N-terminal antibody (arrow). However, no MCEPH band was detected in the immunoprecipitate from mceph/mceph (IP). C. Hippocampi were dissected and an equal amount of lysate from wild type and mceph/mceph was loaded on SDS-PAGE and immunoblotted with anti-Kv1.2. Only a very small fraction of the Kv1.2 was core glycosylated (60 kDa). There appeared to be no increase in the amount of core glycosylated Kv1.2 in mceph/mceph hippocampus compared to wild type. D. HEK293 cells were cotransfected with Kv1.1-DsRed and MCEPH-ZsGreen constructs. Both the 85 kDa Kv1.1-DsRed and the 55 kDa MCEPH-ZsGreen fusion proteins were detected with immunoblotting on cell lysate using the polyclonal Kv1.1 N-terminal antibody (lysate). The relative levels of the two fusion proteins in this overepressing cell system cannot be used to quantitate MCEPH expression or stability in brain since regulation and trafficking is known to be different between these two systems. The lysate was immunoprecipitated with the Kv1.1 C-terminal antibody. In the precipitate, both fusion proteins were detected with the Kv1.1 N-terminal antibody (IP). When the Kv1.1 C-terminal antibody was used for immunoblotting only the Kv1.1-DsRed fusion protein was detected.