Fig. 1. Elements from zizimin1 N-terminal half bind to the CZH2 domain.

A Schematic of zizimin1 structure and fragments used in Figure 1. The Pleckstrin homology (PH), CDM-zizimin homology 1 (CZH1) and CDM-ziziminin homology 2 (CZH2) areas are displayed and amino acid boundaries are indicated. Arrows highlight the three N-terminal areas that interact with the CZH2. Location of HA or Flag tags is indicated by black or white boxes, respectively, gray boxes indicate fragments that are tagged by HA or Flag in different experiments.

B HA-tagged zizimin1 fragments 1–922, 1–539, 534–922 or 923–1515 were expressed in Cos7 cells together with Flag zizimin1_1512-end or Flag zizimin1 PH domain (specificity control) as indicated. 48 hours post transfection, cells were lysed and immunoprecipitated (IPed) with anti-Flag antibody. The IPs and the corresponding whole cell extracts (WCE; representing 9% of the total) were analyzed by Western blotting with anti-HA Ab (top panel) or anti-Flag Ab (bottom panels). Arrows indicate the position of each fragment on the gels, Ab hc is the heavy chain of the Flag Ab used for IP. The PH domain is not visible due to its low molecular weight but expresses at similar levels to the other fragments (see 1C).

C, D. The experiments were done as in B. Ab lc is the light chain of the Flag Ab used for IP. The CZH2 domain associates with the N-terminal subfragments 1–175 and 288–539 but not the PH domain (172–282).

E HA zizimin1_1512-end was expressed in Cos7 cells together with the Flag-tagged minimal CZH1 domain (zizimin1_640–885) or with Flag-PH domain (specificity control) as indicated. Samples were immunoprecipitated with anti-Flag and HA-tagged fragments detected as in B.