Quantification of Tight Junction Proteins

For analysis of the tight junction composition, membrane protein fractions were prepared [23 (link)], and the expression of tight junction proteins discussed as being involved in macromolecule pathway regulation was quantified by immunoblotting employing primary antibodies against tricellulin (rabbit anti-Tric, Invitrogen, Dreireich, Germany, Cat.-No. 700191), occludin (rabbit anti-occludin, Invitrogen, Cat.-No. 711500), angulin-1 (rabbit anti-LSR, Atlas Antibodies, Bromma, Sweden, Cat.-No. HPA007270), and β-Actin (mouse anti-b-Actin, Sigma Aldrich, Cat.-No. A5441) as described [28 (link)]. Specific signals were quantified by chemiluminescence detection after incubation of the washed membranes with Lumi-Light plus (Hoffman La Roche, Basel, Switzerland) using luminescence imaging (Fusion FX7, Vilber Lourmat, Eberhardzell, Germany) and quantification software (Multi-Gauge V2.3, FujiFilm, Tokyo, Japan).