Fig. 5. Yeast two-hybrid interaction analysis using p0071 head 1-198, p0071 head 209-509 and p0071 repeats as baits. (A) Cotransformations of p0071 constructs in pGBKT7 with non-desmosomal cadherin-, dsg and dsc cytoplasmic domains, pg domains, PKP2 and the desmoplakin N-terminus (DP-NTP) were streaked in parallel on plates lacking tryptophane and leucine (-TL) and tryptophane, leucine and histidine (-TLH). Colonies from -TLH plates were blotted on filters and analyzed for lacZ reporter gene activation (lac-Z). The p0071 head 1-198 interacted with dsc3a and pg-3-13+C, but not pg-4-9 or pg-N+1-6. Cotransformants expressing DP-NTP activate the his reporter gene, but not the lac-Z reporter gene. The p0071 head 209-509 interacted with pg-3-13+C and DP-NTP although lac-Z reporter gene activation was weak for the latter interaction. p0071 arm repeats interacted with E-, N-, OB-cadherin/cadherin 11, pg-3-13+C, pg-N+1-6 and PKP2. Again, cotransformants with p0071 repeats and DP-NTP activated the his reporter gene but not the lac-Z reporter gene. E-cadherin interacted with p0071 rep1-5, but not p0071 rep2-10 or p0071 rep4-10, indicating that repeat1 is important for the interaction. (B) Quantitation of lac-Z reporter gene activation by p0071 arm-repeat—cadherin interactions and p0071-head (aa 1-198)—cadherin interactions using ONPG. Lac-Z activity was measured for >5 independent transformants. Lac-Z activation of the N-cadherin-p0071 cotransformants is 3.5-fold higher compared with that of E- and OB-cadherin-p0071 cotransformants.

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