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Fig. 2. Characterisation of BNIP-H and KGA interaction. (A) Pre-immune serum or polyclonal antibodies against BNIP-H were used for immunoprecipitation with mouse brain lysate. The precipitate was analysed by western blotting with antibodies as indicated. Only KGA, but not an unrelated control protein (EEN/endophilin II) coimmunoprecipitated with BNIP-H. Lane 2 shows that there was no nonspecific binding to the protein A/G-beads during the pre-clearing of the brain lysate (see Materials and Methods). (B) Epitope-tagged KGA and BNIP-H expressed in 293T cells were used for co-immunoprecipitation experiments. HA-tagged BNIP-H was coimmunoprecipitated by FLAG-tagged KGA (lane 3). In the reciprocal CoIP, HA-tagged KGA could be coimmunoprecipitated by FLAG-BNIP-H (lane 4) confirming an in vivo interaction between both proteins. HA-KGA did not interact with FLAG-EF1A1 (lane 5). (C,D) Lysates from transfected 293T cells expressing the indicated proteins were immunoprecipitated with an antibody against FLAG, and bound proteins and lysates were analysed with antibodies against FLAG and HA. Schematic representations of the proteins are shown on top. (E) To show direct binding, immobilised beads of GST-BNIP-H, GST-BNIP-2 and GST-BNIP-S were mixed with a FLAG-tagged KGA fragment (aa 134-669) that was produced by in vitro transcription and translation (lane 5). After incubation and washing, the precipitated proteins were analysed by western blotting with FLAG antibody. Only GST-BNIP-H bound to the FLAG-tagged KGA fragment (lane 3), no binding was observed for GST-BNIP-2 or GST-BNIP-S (lane 1 and 2, respectively). No protein was produced in an in vitro transcription and translation reaction without template (lane 4) demonstrating the specificity of the expression system. The amido black staining shows that equal amounts of GST fusion protein were used for this assay. WCL, whole-cell lysates.
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