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. 2012 Apr 24;2:379. doi: 10.1038/srep00379

Figure 1. Identifying 14-3-3γ as a Mieap-binding protein by IP-2DICAL.

Figure 1

(a) Preparation of the samples for identifying endogenous Mieap-interacting proteins by IP-2DICAL. The A549 cells were γ-irradiated, and 40 h after this ionizing radiation (IR), the cell lysates were immunoprecipitated with anti-Mieap antibody (Mieap) or normal rabbit globulin (rIgG), as indicated. The immunoprecipitates were blotted with anti-Mieap antibody. Pre: total cell lysate before IP, Post: total cell lysate after IP, and IP: immunoprecipitated proteins. (b) A two-dimensional display of all (>33,000) the MS peaks in a representative sample prepared as in (a), with the m/z-value (350–1,600 m/z) along the horizontal (x) axis and the LC RT (10–110 min) along the vertical (y) axis. (c) The peak at 540 m/z and 34.9 min displayed in various combinations of axes. The immunoprecipitates generated by the anti-Mieap antibody are indicated in red and the immunoprecipitates from the control rIgG are indicated in blue. Upper left, the m/z and intensity axes, with indicators of the isotopic mass (light blue line and dot). Lower left, a gray-scale intensity pattern of the RT (x axis) and sample (y axis). Upper right, the sample and intensity axes (left) and a box-and-whisker diagram of the immunoprecipitates generated using anti-Mieap antibody and rIgG (right). Lower right, the m/z and RT axes with high (upper) and low (lower) intensities are indicated by a red dot. (d) The interaction of endogenous 14-3-3γ and Mieap proteins. The irradiated A549 cell lysates were generated as in (a) and were subjected to immunoprecipitation with the anti-Mieap antibody (Mieap). Immunoprecipitates generated using rIgG were employed as a control (rIgG). The immunoblot analysis was performed using the anti-14-3-3γ and anti-Mieap antibodies. Lysate: total cell lysate and IP: immunoprecipitated proteins. (e) The interaction of exogenous 14-3-3γ and Mieap proteins. The HCT116 cells were co-transfected with FLAG-tagged 14-3-3γ and HA-tagged Mieap α or β. The transfected cells were γ-irradiated, and 72 h after IR, the cell lysates were immunoprecipitated with anti-FLAG or anti-HA antibodies. Immunoblot analysis was carried out with the anti-FLAG or anti-HA antibodies. IB: immunoblotting, IP: immunoprecipitation, and lysate: total cell lysate.