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Wenxiang Meng, Yoshimi Mushika,Tetsuo Ichii and Masatoshi Takeichi
Adheren junctions (AJ)are protein complexes that occur at cell-cell junctions in epithelial tissues
Beltlike adherens junction that encircles the apical end of an epithelial cell and attaches it to the adjoining cell.
Known to interact with actin, to anchor it to the cell wall. Little is known about it’s interaction it microtubules (MT)
p120-catenin (p120) coupled with cadherin might interact with microtubules (MT) at endogenous levels
This interaction is probably mediated by other proteins
PLEKHA7 (pleckstrin homology domain-containing, family A member 7)
Nehza, a binding partner for PLEKHA7 Associated with minus ends of MTs
KIFC3, kinesin-14 family member concentrated in the ZA Localization abolshed by RNAi of PLEKHA7 or
Nehza
Depletion of any of these proteins led to disorganization of the ZA
Generate GST fusions (Fig 1A)
Incubate with lysates of Caco2
Mass spec materials pulled down, found PLEKHA7
To confirm finding, FLAG tag PLEKHA7, transfect Caco2
Incubate transfected lysates with GST fusions and GST , probe for FLAG (Fig 1C)
IP with α-FLAG, p120 and E-cadherin CO-IP (Fig 1D)
Further analysis shows region between aa 538 – 696 in PLEKHA7 is required to bind to p120
Developed Abs for PLEKHA7
Immunofluorescence staining of Caco2
Examine the effect of p120 depletion on PLEKHA7 by siRNA
Look at PLEKHA7 recruitment in Neuro-2a cells (cadherin-deficient)
overexpress PLEKHA7-GFP, see what happens
siRNA of PLEKHA-7
PLEKHA7 is important for cadherin-catenin accumulation at the ZA
Use GST-PLEKHA7 fusion to pull down Nehza
Developed Abs for Nehza
Transfect Caco2 w/ PLEKHA-FLAG, and Nehza-GFP
Use transfected cell lysates to see if they
CO-IP
Deletion analysis of Nehza shows C-terminal region from aa 943 – 1225 is required for association with PLEKHA7
Look at fluorescence to see where it localizes (Fig2D) Compare to PLEKHA7-
siRNA knockdowns
Examine localization of PLEKHA7 when Nehza is knockdown by siRNA
Nehza, once associated with PLEKHA7, maintains the integrity of the ZA
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