GoKawiilCell lines
HT29 (HTB-38), T84 (CCL-248) and 293T (CRL-3216) cells were originally obtained from the ATCC. HT29-Cas9 cells were generated as previously described21. All cell lines were grown at 37 °C and 5% CO 2 . HT29 cell lines were propagated in McCoy’s 5A medium, T84 lines in DMEM/F-12 and 293T cells in DMEM; all supplemented with 10% FBS. Cell lines were routinely tested for mycoplasma contamination and found to be negative.
Antibodies for immunoblotting, immunoprecipitation and flow cytometry
The following mouse monoclonal antibodies were purchased from the indicated vendors and used at the indicated dilutions: β-actin (A5441, Sigma, 1:2,000), claudin-4 (sc-376643, Santa Cruz Biotechnology, 1:200), Alexa Fluor 647-conjugated claudin-4 (FAB4219R, R&D, 1:100), His tag (MAB050, R&D Systems, 5 μg ml−1), GFP (MA1-952, Invitrogen, 1:2,000). The following rabbit monoclonal antibodies were purchased from the indicated vendors: claudin-3 (83609S, Cell Signaling Technology, 1:1,000), Na/K ATPase α1 (D4Y7E, Cell Signaling Technology, 1:1,000). The following rat monoclonal antibodies were purchased from the indicated vendors: E-cadherin ectodomain (14-3249-82, Invitrogen, 1:250), PE-conjugated E-cadherin ectodomain (147304, BioLegend, 1:100). The following rabbit polyclonal antibodies were purchased from the indicated vendors: claudin-4 (36-4800, Invitrogen; see ‘Co-immunoprecipitation’, Methods), claudin-2 (51-6100, Invitrogen; see ‘Co-immunoprecipitation’, Methods). Rabbit polyclonal antiserum against BFT was produced as previously described61 and was used at 1:1,000. The following secondary antibodies were purchased from the indicated vendors: Alexa Fluor 488-conjugated anti-mouse IgG (A-11001, Invitrogen, 5 μg ml−1), HRP-conjugated anti-rabbit IgG (7074, Cell Signaling Technology, 1:1,000), HRP-conjugated anti-rat IgG (7077, Cell Signaling Technology, 1:1,000), HRP-conjugated anti-mouse IgG (R1005, Kindle Biosciences, 1:1,000).
Plasmids and molecular cloning
The plasmids GFP-C4 (pCMV::EGFP-hCLDN4) and GFP-claudin-2 (pCMV::EGFP-hCLDN2) expressing N-terminally EGFP-tagged human claudins from the CMV promoter were obtained as a gift from J. Turner (Harvard Medical School).
The plasmids GFP-C3 (pCMV::EGFP-hCLDN3), GFP-claudin-4(ΔCTD) (pCMV::EGFP-hCLDN4ΔCTD), GFP-claudin-4(ECS1 3 ) (pCMV::EGFP-hCLDN4ECS1-3), GFP-claudin-4(ECS2 3 ) (pCMV::EGFP-hCLDN4ECS2-3) and Ecad-mCherry (pCMV::hCDH1-mCherry) were constructed using In-Fusion cloning (Takara Bio). GFP-C3 was generated by replacing the claudin-4 sequence of GFP-C4 with human claudin-3 complementary DNA. GFP-claudin-4(ΔCTD) was generated by excising the claudin-4 amino acids Asn184–Val209 from GFP-C4. GFP-claudin-4(ECS1 3 ) was generated by replacing the claudin-4 amino acids Met29–Arg81 from GFP-C4 with the claudin-3 amino acids Met28–Arg80. GFP-claudin-4(ECS2 3 ) was generated by replacing the claudin-4 amino acids Thr139–Met160 from GFP-C4 with the claudin-3 amino acids Ser138–Met159. Ecad-mCherry was generated by replacing the mouse E-cadherin sequence from the plasmid Murine E-cadherin mCherry (Addgene, 71366) with the human E-cadherin sequence from the plasmid E-cadherin-GFP (Addgene, 28009). The plasmids GFP-claudin-4(T33S) (pCMV::EGFP-hCLDN4T33S), GFP-claudin-4(V41I) (pCMV::EGFP-hCLDN4V41I) and GFP-claudin-4(T45N) (pCMV::EGFP-hCLDN4T45N) were constructed using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies).
Recombinant proteins
His 6 -tagged BFT
The BFT-2 isoform (herein referred to as BFT) was used for all experiments. Recombinant proBFT (residues Asp24–Asp397) with a C-terminal His 6 -tag was expressed from a modified pET30a vector in Escherichia coli Rosetta(DE3). Overnight cultures of E. coli were subcultured at a ratio of 1:50 into 100 ml of lysogeny broth with kanamycin and chloramphenicol, grown at 37 °C and 240 rpm until they reached an optical density (OD) 600 of 0.4–0.6 and then induced with 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at room temperature overnight. The following morning, cells were collected by centrifugation at 3,000g for 15 min at 4 °C. Pellets were resuspended in ice-cold buffer A (50 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, pH 7.5) and sonicated to lyse cells. Cell debris was pelleted by centrifugation at 17,200g at 4 °C for 45 min. The supernatant containing recombinant proBFT was passed through a 0.22-μm filter and loaded onto a HisTrap column (Cytiva Life Sciences) pre-equilibrated with 10 column-volumes (CV) of buffer A. The column was washed with 5 CV of buffer A, then protein was eluted with 5 CV of buffer A containing 250 mM imidazole. Fractions containing the A 280 peak were pooled and dialysed into PBS overnight. The resulting proBFT was trypsinized (T6567, Sigma) at a 1:30 w/w ratio of trypsin:proBFT to liberate mature BFT, which was subsequently re-purified on a HisTrap column and dialysed into PBS overnight as above. Concentration was determined by Qubit protein assay (Pierce), purity was assessed by SDS–PAGE followed by Coomassie blue staining (Extended Data Fig. 1a) and purified BFT was aliquoted and stored at −80 °C until use.
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