ARF4 is not required for ciliogenesis in vivo or in vitro, but its loss results in polydactyly. (A) Images of differentiating keratinocytes transduced with scrambled control, Arf4, or Bbs2 shRNAs and then immunolabeled with antibodies against acetylated tubulin to visualize stable microtubules, including primary cilia. See magnified examples in the panels at the right, showing that cilia still form upon depletion of either of these proteins. Bars: 15 µm; (high-magnification cilia) 1 µm. (B) Single confocal plane of E15.5 WM epidermis immunolabeled with an antibody to acetylated tubulin (green). H2B-RFP (red) marks transduced cells. Boxed image at left is shown magnified at right. Arrows point to primary cilia. Bars, 15 μm. (C) Image of E17.5 embryo transduced with Arf4-2 shRNA, showing examples of polydactyly, observed in 75% of ARF4 KD embryos examined. Bars, 1 mm.