Localisation of MarvelD3 by epifluorescence microscopy. (A, B) Immunostaining of Caco-2 (A) and HCE (B) cells with anti-MarvelD3 and anti-occludin antibodies. Cells were cultured on glass coverslips and fixed with methanol. (C) Localisation of MarvelD3 constructs in Caco-2 and MDCK cells. Caco-2 and MDCK cells were transfected with full length constructs of MarvelD3 isoform 1 (MD3_1) and isoform 2 (MD3_2). The samples were then fixed with methanol and labelled with the anti-MarvelD3 antibody. Note, both isoforms are equally distributed and localise to cell junctions. (D) Expression of HA-tagged MarvelD3. Caco-2 cells were transfected with HA-tagged variants of the MarvelD3 isoforms and then double labelled with anti-HA and anti-MarvelD3 antibodies. Bars, 10 μm.