Depletion of ARF4 results in intracellular accumulation of Notch signaling components in vitro and in vivo and reduced HES1 expression in developing epidermis. (A) Differentiating cultured keratinocytes infected with LV harboring Arf4-2 or scrambled control shRNA and then immunolabeled for Presenilin-2 (red). DAPI marks nuclei. Arrowheads point to perinuclear vesicular accumulation of presenlin-2 upon depletion of Arf4. (B) Quantification of Presenilin-2 basal body localization in Arf4 KD keratinocytes. Data in histogram are from two independent experiments in which 100–200 cells were analyzed per condition. (C) Differentiating cultured keratinocytes transduced with Arf4 shRNA and immunolabeled for Notch 2 (gray) and GM130 (cyan) to visualize Golgi. H2B-RFP marks transduced cells (red) versus nontransduced control cells. (D) Quantification of Notch 2-Golgi fluorescence in Arf4 versus scrambled control shRNA-expressing keratinocytes. Data in histogram are from two independent experiments in which 100–200 cells were analyzed per condition. (E) Notch 2 (green) expression in Arf4-2 shRNA (inset, H2B-RFP nuclei) transduced tissue versus uninfected littermate control. DAPI marks nuclei. Boxed regions are magnified in the images at right. Arrows point to perinuclear accumulation of Notch. (F) Expression of HES1, a downstream Notch target gene, is no longer seen in the suprabasal layers of epidermis that has been depleted of the small GTPase ARF4. Bars: (A–C) 15 µm; (E and F) 30 µm.