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NAPA-1 is the critical region for trans-complementation.

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posted on 03.01.2012, 02:12 authored by Jingfen Han, Diego Miranda-Saavedra, Nathan Luebbering, Aman Singh, Gary Sibbet, Michael A. J. Ferguson, Vaughn Cleghon

The N-terminal DmDYRK2 molecules shown schematically in (A) were assessed for their ability to complement activation loop phosphorylation by ΔN2-DmDYRK2. All proteins contain a FLAG epitope. The presence and location of the conserved NAPA-1, NAPA-2, and DH box regions within the truncated proteins are indicated. (B) ΔN2-DmDYRK2 was expressed alone in Sf9 cells or it was co-expressed with N-terminal truncated proteins as indicated. WT- and kinase-inactive K227M-DmDYRK2 proteins were included as controls. In the top two panels of B, DmDYRK2 proteins containing the kinase domain were immunoprecipitated from cell lysates with anti-DmDYRK2 antibody, fractionated by SDS/PAGE and analyzed on immunoblots. Levels of proteins were compared by probing blots with anti-FLAG antibody (α-FLAG) and levels of activation loop phosphorylation were assessed with anti-pTyr antibody (α-pTyr). To confirm that all proteins were expressed, Sf9 cell lysates were fractionated by SDS/PAGE and the levels of the DmDYRK2 proteins determined by probing with anti-FLAG antibody (α-FLAG Lysates). The experiments shown were performed at least three separate times.