Journal: bioRxiv

Article Title: Angulin-1 seals tricellular contacts independently of tricellulin and claudins

doi: 10.1101/2020.10.02.323378

Figure Lengend Snippet: (A) Lysates of angulin-1 KO cells were incubated with GST and GST-fusion proteins of aa 409-575 and aa 409-570 of angulin-1 (GST-ang575 and GST-ang570, respectively), and subjected to GST pull-down assays. The lysates (input) and precipitates with GST, GST-ang575, and GST-ang570 were analyzed by western blotting with anti-ZO-1 mAb, anti-ZO-2 pAb, and anti-ZO-3 pAb. The precipitates were also immunoblotted with anti-GST pAb. (B) Schematic diagram of the domain structure of mouse ZO-1 protein with 1745 amino acids. ZO-1 contains three PDZ domains, an SH3 domain, a guanylate kinase domain (GUK), and an acidic domain (AD) in its N-terminal half. Four distinct portions of ZO-1 indicated as solid lines with amino acid numbers were produced as recombinant fusion proteins with MBP. (C) GST pull-down assays of MBP-N-ZO-1 fusion protein with GST-ang575. Input: crude lysate of Escherichia coli expressing MBP-N-ZO-1. (D) GST pull-down assays of MBP-fusion proteins of PDZ1, PDZ2, and PDZ3 of ZO-1 (MBP-zPDZ1, MBP-zPDZ2, and MBP-zPDZ3, respectively) with GST-ang575. Input: each purified MBP-fusion protein. In (C) and (D), the samples were subjected to SDS-PAGE followed by Coomassie Brilliant Blue (CBB) staining. (E) Schematic diagram of a chimeric protein of angulin-1 Δpbm and ZO-1 (angulin-1 Δpbm-ZO-1). (F) Western blotting of lysates of MDCK II cells, mock-transfected angulin-1 KO cells, and angulin-1 KO cells expressing angulin-1 Δpbm or angulin-1 Δpbm-ZO-1 with anti-angulin-1 pAb or anti-α-tubulin mAb. (G) Angulin-1 KO cells expressing angulin-1 Δpbm-ZO-1 were immunostained with anti-angulin-1 mAb and anti-claudin-2 mAb. Confocal sections of the apical region including claudin-2 staining and the lateral region together with the corresponding Z-stack images along the white dotted lines are shown. Arrowheads indicate TCs. (H) Paracellular flux of fluorescein in MDCK II cells, angulin-1 KO cells, two independent mock-transfected angulin-1 KO cell clones (+mock_1 and +mock_2), and two independent angulin-1 KO cell clones expressing exogenous angulin-1 Δpbm (+angulin-1Δpbm_1 and +angulin-1Δpbm_2). Data are shown as mean ± SD ( n =3) and were analyzed by the Tukey–Kramer test. * P <0.01. The data in MDCK II cells, angulin-1 KO cells, and two independent mock-transfected angulin-1 KO cell clones are identical to those in . (I) EM observation of horizontal ultrathin sections of angulin-1 KO cells expressing exogenous angulin-1 Δpbm. TCs at the level of TJs (TJ level) and the more basal level of the lateral membrane containing desmosomes (DS level) were analyzed. DS: desmosome. Bars: 10 μm (G) and 200 nm (I).

Article Snippet: Mouse anti-claudin-2 mAb (32-5600), rabbit anti-tricellulin pAb (48-8400), rabbit anti-ZO-2 pAb (38-9100) and rabbit anti-ZO-3 pAb (36-4100) were purchased from Thermo Fisher Scientific.

Techniques: Incubation, Western Blot, Produced, Recombinant, Expressing, Purification, SDS Page, Staining, Transfection, Clone Assay