FIGURE 5.
Effect of substitutions in Cld3 on binding of cCPE and mutants thereof. HEK cells transfected with Cld3WT or Cld3 mutant were used for pulldown (A and B) or cellular binding assays (C–E). For pulldown assays, representative blots (A) and quantification (B) are shown. Cld3N148D interacts with GST-cCPEWT more weakly than Cld3WT, whereas R157Y in Cld3 does not affect binding. Results are mean ± S.E. (error bars); n = 4. *, p < 0.05 to Cld3WT. C, cellular binding assays analyzed by Western blotting verified inhibition of cCPE binding by N148D but not R157Y (left panel). N148D in Cld3 blocks binding of cCPE mutants (middle left panel) whereas R157Y does not enhance the effects of substitutions in cCPE (middle right panel). D and E, cellular binding assays were analyzed using a fluorescence plate reader. GST-cCPEWT binds to Cld3K156A as well as to Cld3WT. D, additive effect of changed charge in ECL2 of Cld3 (E153V, K156A, R157Y) and substitutions in cCPE was tested. E, binding of GST-cCPE mutants to Cld3 construct was quantified relative to GST-cCPEWT binding (relative binding). Substitutions K156A and R157Y do not change the effect of substitutions in cCPE considerably. E153V slightly decrease relative binding of GST-cCPEY306A. Results show mean ± S.E. (error bars); n ≥4). *, p < 0.05 to GST-cCPEWT.