Homotropic dimerization of NaPi-IIa.A, MYTH 2.0 dot assays.

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Homotropic dimerization of NaPi-IIa.A, MYTH 2.0 dot assays. Homotropic dimerization of NaPi-IIa.A, MYTH 2.0 dot assays. TF-Cub-Baits in pTLB-1 harboring full-length NaPi-IIa, its C-terminal truncation for TRL (ΔTRL) or the entire tail (ΔCT), and full-length NHE-1 as a negative control were co-transformed in a yeast reporter strain with either NubG-HA-Preys of NaPi-IIa, NHE-3, Ost1-NubG, or Ost1-NubI. Prototrophic cells were rescued on minimal SD−Trp/−Leu/−Ade/−His (SD−WLAH) plates supplemented with 3-AT to suppress leakage of the respective baits. Rescued colonies were blue due to induction of lacZ as determined by β-galactosidase replica colony lift experiments (not shown). B, immunoprecipitation from total membranes of X. laevis oocytes. Various cRNAs (MYC-NaPi-IIa, its corresponding TRL ablation, or MYC-NaS1) were co-injected with cRNA of HA-NaPi-IIa in oocytes. Total solubilized membranes of oocytes were processed for immunoprecipitation by anti-HA. The immunocomplexes were resolved by SDS-PAGE for immunoblotting with the antibodies shown. Results from two other experiments were identical (n = 3). The detection of type IIa homotropic dimers was abrogated when solubilized membranes of oocytes after a single injection of HA-NaPi-IIa were mixed with those of MYC-NaPi-IIa-injected oocytes and used for immunoprecipitation (IP). PM, plasma membrane; SD−WL, SD−Trp/−Leu. Serge M. Gisler et al. Mol Cell Proteomics 2008;7:1362-1377 © 2008 The American Society for Biochemistry and Molecular Biology