Anti human zo 1 mab

After the indicated culture period on Transwells, cells were fixed with 4 % paraformaldehyde for 30 min at 37 °C. Anti-human ZO-1 mAb (Zymed Laboratories Inc., San Francisco, CA), anti-human E-cad rabbit mAb (Cell Signaling Technology, MA), or anti-human PCDH1 mAb (Santa Cruz) was used as a primary antibody, and Alexa 488-conjugated anti-mouse IgG was used as a secondary antibody. An FV1000-D laser scanning confocal microscope with a 60× objective lens was used to investigate expression.

Stimulated cells were washed twice with ice-cold PBS and lysed in Tris-buffered saline containing 1 % Nonidet P-40, 60 mM octyl-β-glucoside, 2 mM phenylmethylsulfonylfluoride, 10 μg/ml aprotinin, 2 μg/ml leupeptin and pepstatin A, 50 mM NaF, and 1 mM sodium orthovanadate for 30 min on ice. The lysates or immunoprecipitates were centrifuged for 15 min at 14000 g. The samples for polyacrylamide gel electrophoresis (PAGE) analysis were mixed with 4× XT sample buffer (Bio-Rad, Hercules, CA) and boiled for 4 min and separated on 10 % sodium dodecylsulfate-PAGE and transferred onto an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated with anti-human ZO-1 mAb (Zymed), anti-human OCLN rabbit mAb (Zymed), anti-human E-cad rabbit mAb (Cell Signaling), and anti-human PCDH1 mAb (Santa Cruz) as a primary antibody and an appropriate secondary horseradish peroxidase-conjugated antibody (Fig. 6). Signals were detected using enhanced chemiluminescence (GE Healthcare, Little Chalfont, UK).