Tissue distribution and subcellular localization of Unc5CL. (A) Unc5CL-specific western blot of whole tissue lysates from the indicated tissues or overexpressed untagged Unc5CL as marker. (B) Relative Unc5CL mRNA expression in epithelial cell preparations from consecutive small intestinal (1–6) or colonic segments. Data represent the mean values ±S.D. of technical triplicates. (C) Unc5CL-specific western blot of intestinal (1–6) and colonic epithelial samples shown in (B). (D) Unc5CL-specific western blot of lysates from CaCo-2 cells stably expressing mock (scr sh) or Unc5CL-specific shRNAs (sh1–sh5). (E) Proteins from CaCo-2 cells were subjected to TX-114 phase separation. Fractions were analyzed by western blot using the indicated antibodies. CytoC, 14 kDa Cytochrome C, I, detergent-insoluble proteins, D, detergent phase, amphiphilic integral membrane proteins, A, aqueous phase, hydrophilic proteins. (F) z-stack reconstruction following confocal microscopy of overexpressed C-terminally FLAG-tagged Unc5CL (Unc5CL) or Unc5CL lacking the transmembrane domain (ΔTM) in CaCo-2 cells using FLAG-specific antibodies. (G) (a–c) Immunofluorescent staining of Unc5CL on cryosections from OCT embedded uterus using Unc5CL-specific antibodies. Blue: nuclear DAPI staining. (a, b) Green: Unc5CL, (c) isotype control (FLAG). (A, D and E) Asterisks mark unspecific bands