Tight Junction Protein Localization in MDCK Cells

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.