FIGURE 4.

FKBP38 is a negative regulator of Shh signal transduction. A, confluent cultures of Fkbp38^+/+ or Fkbp38^−/− MEFs that had been deprived of serum for 24 h by incubation in Opti-MEM reduced serum medium were fixed and processed for immunofluorescence analysis with antibodies to γ-tubulin and to acetylated α-tubulin. Nuclei were also stained with Hoechst 33258, and the cells were then examined with a confocal microscope. Merged images are also shown; scale bars, 20 μm. Higher magnification views are boxed; scale bars, 5 μm. B, Fkbp38^+/+ and Fkbp38^−/− MEFs were incubated in the absence or presence of Shh for 48 h, after which whole cell homogenates were prepared and subjected to immunoblot analysis (IB) with anti-Gli1, anti-FKBP38, anti-ANKMY2, and anti-HSP90. C, Fkbp38^+/+ and Fkbp38^−/− MEFs were incubated in the absence or presence of Shh for 24 h, after which total RNA was extracted from the cells and subjected to RT and real-time PCR analysis of Gli1 and Ptch1 mRNAs. Data are presented as relative -fold induction by Shh and are means ± S.D. from five independent experiments. **, p < 0.01 versus the corresponding value for Fkbp38^+/+ cells (Student's t test).