FIGURE 1.
Identification of FKBP38-associated proteins by proteomics analysis of transgenic mice. A, whole brain lysates prepared from WT or six different lines of Fkbp38TG (harboring one allele of a His6-FLAG-tagged FKBP38 transgene) mice were subjected to immunoblot (IB) analysis with anti-FLAG, anti-FKBP38, and anti-calnexin (loading control). B, gross appearance of newborns of Fkbp38−/− (left) and Fkbp38−/−;TG (right) mice. Arrows, neural tube closure defects; arrowheads, normal neural tube closure. Scale bar, 5 mm. C, extracts of WT or Fkbp38TG mouse brain were subjected to dual affinity chromatography with anti-FLAG and Ni-NTA resin. The purified proteins were fractionated by SDS-PAGE and stained with silver. The arrowhead indicates the band corresponding to His6-FLAG-FKBP38. D, immunoblot analysis of various mouse tissues with anti-ANKMY2, anti-FKBP38, and anti-HSP90 (loading control). Asterisks, nonspecific bands; arrowheads, corresponding specific bands. E, lysates prepared from Fkbp38−/−, Fkbp38+/+, and Fkbp38+/− mouse embryos (E11.5) were subjected to immunoblot analysis with anti-ANKMY2, anti-FKBP38, and anti-HSP90. F, immunohistofluorescence analysis of a neural tube section from a WT mouse embryo (E11.5) with anti-ANKMY2. Nuclei were stained with Hoechst 33258. Dashed line, neural tube. FP, floor plate. Scale bar, 100 μm. G, extracts of WT or Fkbp38TG mouse brain were subjected to immunoprecipitation (IP) with anti-FLAG. The resulting precipitates, as well as a portion (5% of the input for immunoprecipitation) of the extracts, were subjected to immunoblot analysis with anti-ANKMY2, anti-FLAG, and anti-HSP90. H, WT mouse brain extract was subjected to immunoprecipitation with anti-ANKMY2, anti-FKBP38, or control rabbit IgG, after which the resulting precipitates, as well as a portion (3% of the input for immunoprecipitation) of the tissue extract, were subjected to immunoblot analysis with anti-FKBP38 and anti-ANKMY2. Asterisk and arrowhead, nonspecific and specific bands, respectively.