Presenilin-2 localizes to basal bodies via a VxPx trafficking module in vitro and in vivo. (A) 1°MKs were induced to differentiate in 2 mM Ca²⁺ and then subjected to IF microscopy with antibodies against Presenilin-2 (red) and γ-tubulin (green). DAPI marks nuclei and arrowheads point to colocalization of Presenilin-2 and γ-tubulin. (B) 1°MKs depleted of Presenilin-2 and immunolabeled for antibodies against Presenilin-2 (to verify KD) or acetylated microtubules (MTs) to visualize cilia (and other stable microtubules within the cell). Arrows point to cilia. (C) Western blot analysis of cell lysates from 1°MKs depleted of Presenilin-2 with one of three different shRNAs and then probed with antibodies against Presenilin-2 and NICD1. Tubulin was used as a loading control (not depicted). (D) Schematic of the LV-mediated expression constructs used for shRNA KD and rescue. High-titer viruses were used for injection into the amniotic sacs of E9.5 embryos as described in the text. (E) Western blots of lysates from keratinocytes transduced with Psen1,2 shRNA LV and probed with antibodies against Presenilin-1, Presenilin-2, and tubulin (shown as loading control). (F and G) Expression of LV WT GFP-Presenilin-2 or P486A (VxPx mutant) GFP-Presenilin-2 in differentiating cultured keratinocytes, colabeled with γ-tubulin (blue) to visualize basal bodies/centrosomes (arrows). Merged image shows junctional and basal body colocalization of GFP-Presenilin-2 (green) and γ-tubulin (blue). Note that in contrast, the P486A mutant only localized to cell junctions (better visualized in J). Boxed region shown in panels at left are magnified in the two panels at right, and arrows point to basal bodies. (H) Localization of ARL13b (red) and WT GFP-Presenilin-2 (green) in E15.5 epidermis. High-magnification image from boxed region shows example of cilia or basal body localization of GFP-PRESENILIN (arrowhead). (I) ARL13b (red) at nascent WT GFP-Presenilin-2 (+) cell junctions in stratifiying E15.5 epidermis. (J) Single confocal plane from E15.5 WM epidermis transduced with Psen1,2 shRNA and either WT Presenilin-2 GFP or P486A Presenilin-2 GFP, and arrows point to basal body. Bars, 15 μm. (K) Quantification of basal body localization of WT or P486A Presenilin-2; quantification represents data from WM epidermis from at least three different embryos. Error bars are standard deviation. *, P < 0.001, Student’s t test.