ZO proteins (ZO-1, ZO-2, ZO-3) are scaffold proteins that are concentrated at the cytoplasmic surfaces of the junctional complexes and determine the specialization and localization of the junction. ZOs form the plaque structures underlying plasma membranes together with proteins such as cingulin and symplekin. All three ZO proteins have three PDZ domains which characterize them as membrane-associated guanylate kinase-like homologues (MAGUKs).
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Immunofluorescent detection of Zo-1 in MDCK cells. Confluent monolayers were fixed in 50% methanol/50% Acetone, blocked for at least 30 minutes in 1% BSA then incubated 2 hours with a Zo-1 monoclonal antibody (Cat. No. 33-9100) at 5 µg/mL, washed, then incubated 1 hour with Alexa Fluor 488 conjugated Donkey anti-Mouse secondary antibody (Cat. No. A-21202) at a dilution of 1:2000. Cells were counterstained with DAPI (blue). Coverslips were mounted with Prolong Gold Antifade reagent (Cat. No. P36930) and imaged at 40X. Images generated by Joell Solan in Paul Lampe Lab at the Fred Hutchinson cancer Research Center.
Western blot analysis of Zona Occludens protein 2 (ZO-2). Performed by loading 50 µg of MDCK (lane 2) and NRK (lane 3) cell lysates and 2 µL SeeBlue Plus2 Prestained Protein Ladder (Cat. No. LC5925) (lane 1) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 1% BSA/TBST for at least 1 hour at room temperature. ZO-2 was detected using a ZO-2 polyclonal antibody (Cat. No. 38-9100) at a concentration of 1 µg/mL in blocking buffer overnight at 4°C on a rocking platform, followed by a goat anti-rabbit IgG Alexa Fluor 680 conjugated secondary antibody (Cat. No. A-21109) at a dilution of 1:10,000 for at least 1 hour. Fluorescent detection was performed using the Odyssey® CLx imaging system (Li-cor Biosciences).
Immunohistochemistry analysis of ZO-3/TJP3. Staining in the membrane of paraffin-embedded human colon tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 minutes. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 minutes at room temperature, washed with ddH2O and PBS, and then probed with a ZO-3/TJP3 polyclonal antibody (Cat. No. 36-4000) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.