Fig. 6: IFT20 interacts with Ceramide-PKCζ signaling complex, and deletion of IFT20 inhibits the expression and apical localization of β-catenin in the osteoblasts.

The immunofluorescent images captured using Leica DMI6000 inverted epifluorescence microscope under 40X lens of the spheroidal cryosections were z-stacked and 3D-deconvolutionarily processed by LAS-X (Leica) acquisition software. Immunofluorescence staining of Ceramide (a, g) and IFT20 (b, h) in the cryosections revealed the colocalization of the molecules (c, i). Immunofluorescence staining of phosphorylated PKCζ (pPKCζ) (d, j) and IFT20 (e, k) in the cryosections revealed the colocalization of the molecules (f, l). Immunoprecipitation using anti-Ceramide antibodies (Sigma-Aldrich C8104–50TST) were performed with protein lysates prepared from Ad-null infected or Ad-cre infected osteoblasts and Western for anti-PARD3, anti-pPKCζ, anti-PKCζ and anti-IFT20 antibodies (m). Western analysis of IFT20, pGSK3β, total GSK3β, pPKCζ, total PKCζ, β-catenin, α-catenin, and β-actin was performed (n). Quantification of protein levels of IFT20, pPKCζ, PKCζ, β-catenin and α-catenin was obtained by imageJ plot profile function. The intensity of the bands was averaged from 3 different experiments (o). The tissue slides of cryosection focusing on the endosteal surface of cortical bone from the tamoxifen treated IFT20f/f control mice (p, r) and IFT20f/f;Col1cre-ERT mice (q, s) were subjected to the immunofluorescence study using anti-β-catenin antibodies and Rhodamine phalloidin. Micrographs representing Z-stacked 3D-deconvolution processed images captured using Leica DMI6000 inverted epifluorescence microscope under ×40 lens (p–s). Yellow arrow points to the relatively long primary cilia. Scale bar (a–f, p–s) 10 μm, scale bar (g–l), 5 μm.