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4 protocols using mouse anti e cadherin mab eccd 2 m108

1

MDCK II Cell Culture and Tight Junction Characterization

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MDCK II cells were provided by Dr. Masayuki Murata [9 (link)]. Cells were grown in DMEM (high glucose) supplemented with 5% fetal bovine serum. Mouse anti-ZO-1 monoclonal antibody (mAb) (T8/754), rat anti-occludin mAb (MOC37), rabbit anti-claudin-2 polyclonal antibody (pAb), and rabbit anti-claudin-4 pAb were characterized as described previously [27 (link)–30 (link)]. Rabbit anti-claudin-1 pAb (51–9000), mouse anti-claudin-2 mAb (32–5600), rabbit anti-claudin-3 pAb (34–1700), mouse anti-claudin-4 mAb (32–9400), rabbit anti-claudin-7 pAb (34–9100), and alexa fluor 488 phalloidin (A12379) were purchased from Invitrogen. Rabbit anti-nonmuscle myosin heavy chain II-B (MHC-B) pAb (PRB-445P) was purchased from Covance. Mouse anti-E-cadherin mAb (ECCD-2; M108) was purchased from Clontech. Fluorescein isothiocyanate-dextran (FITC-dextran), bumetanide (B3023) and NPPB (N4779) were purchased from Sigma-Aldrich.
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2

Tight Junction Protein Localization in MDCK Cells

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MDCK II cells were provided by Dr. Masayuki Murata [9 (link)]. MDCK I cells were provided by the late Dr. Shoichiro Tsukita (Kyoto University) and maintained in our laboratory [9 (link)]. Cells were grown in DMEM (high glucose) supplemented with 5% fetal bovine serum.
Mouse anti-ZO-1 monoclonal antibody (mAb) (T8/754), rat anti-occludin mAb (MOC37), rabbit anti-claudin-2 polyclonal antibody (pAb), and rabbit anti-claudin-4 pAb were characterized as described previously [5 (link),22 (link)–24 (link)]. Rabbit anti-ZO-2 pAb (38–9100), rabbit anti-ZO-3 pAb (36–4100), rabbit anti-claudin-1 pAb (51–9000), mouse anti-claudin-2 mAb (32–5600), rabbit anti-claudin-3 pAb (34–1700), mouse anti-claudin-4 mAb (32–9400), rabbit anti-claudin-7 pAb (34–9100), and alexa fluor 488 phalloidin (A12379) were purchased from Invitrogen. Rabbit anti-FLAG pAb (PM020) and rat anti-GFP mAb (D153–3) were purchased from Medical and Biological Laboratories. Mouse anti-FLAG mAb (018–22381) was purchased from Wako. Rabbit anti-nonmuscle myosin heavy chain II-B (MHC-B) pAb (PRB-445P) was purchased from Covance. Mouse anti-E-cadherin mAb (ECCD-2; M108) was purchased from Clontech. Fluorescein isothiocyanate-dextran (FITC-dextran) was purchased from Sigma-Aldrich. Fluorescein (16106–82) was purchased from Nacalai tesque.
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3

Characterization of Tight Junction Protein Antibodies

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Mouse anti-ZO-1 monoclonal antibody (mAb) (T8/754), rat anti-occludin mAb (MOC37), and rabbit anti-claudin-2 polyclonal antibody (pAb) were characterized as described previously [41 (link)–43 (link)]. Mouse anti-claudin-2 mAb (32–5600), rabbit anti-claudin-3 pAb (34–1700), alexa fluor 488 phalloidin (A12379) and alexa fluor 647 phalloidin (A22287) were purchased from Life Technologies. Mouse anti-E-cadherin mAb (ECCD-2; M108) was purchased from Clontech. Mouse anti-Na+/K+ ATPase α1 mAb (NB300-146) was purchased from Novus Biologicals. Fluorescein isothiocyanate-dextran (FITC-dextran), benzamil (B2417), bumetanide (B3023), ouabain (O3125), H89 (B1427), genistein (G6649), Go6983 (G1918) and 3-isobutyl-1-methylxanthine (IBMX, I5879) were purchased from Sigma Aldrich. Fluorescein (16106–82) was purchased from Nacalai tesque. SQ22536 was purchased from Santa Cruz. Forskolin was purchased from Wako.
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4

Characterization of MDCK Cell Tight Junctions

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The MDCK II cells were provided by Dr. Masayuki Murata [19 (link)]. MDCK I cells were provided by the late Dr. Shoichiro Tsukita (Kyoto University) and maintained in our laboratory [19 (link)]. Cells were grown in DMEM (high glucose) supplemented with 5% fetal bovine serum. Electrophysiological and morphological experiments were performed on cells cultured for 6 days on 12-mm-diameter Transwell filter inserts with a 0.4-μm pore size (Corning, Corning, NY) unless otherwise noted. The cells were plated at a density of 2 × 105 cells/cm2 on filter inserts, and the culture medium was exchanged every day.
Mouse anti-ZO-1 monoclonal antibody (mAb) (T8/754), rat anti-occludin mAb (MOC37), and rabbit anti-claudin-2 polyclonal antibody (pAb) were characterized as described previously [50 (link)–52 (link)]. Mouse anti-claudin-2 mAb (32–5600), rabbit anti-claudin-3 pAb (34–1700), and Alexa Fluor 488 phalloidin (A12379) were purchased from Invitrogen. The rabbit anti-nonmuscle myosin heavy chain II-B (MHC-B) pAb (PRB-445P) was obtained from Covance. The mouse anti-E-cadherin mAb (ECCD-2; M108) was purchased from Clontech.
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