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Fig. 3. Rat Nedd4 domains WW1, WW2 and WW3 bind to Cx43. The first lanes were loaded with cell lysates of WB-F344 cells as a Cx43 size control. As negative controls for the pull-down analyses GST without any fusion protein was used. Further negative controls were samples of the different GST-WW fusion proteins that were incubated with buffer (-) instead of WB-F344 cell lysates (+). (A) SDS-PAGE and immunoblot of Cx43 obtained in pull-down assays with GST-fusion proteins of rat Nedd4 domains WW1, WW2 and WW3. Cx43 was recognized by anti-Cx43 antibody 71-0700, which detects non-phosphorylated and several phosphorylated forms of Cx43. Upper panel: in pull-down experiments with cell lysates from untreated WB-F344 cells WW1, WW2 and WW3 of rNedd4 bound to rCx43. As indicated by arrows, WW1 and WW2 mainly bound the non-phosphorylated form of Cx43 (P0), whereas domain WW3 interacted with the non-phosphorylated form of Cx43 (P0) and the P1-isoform of Cx43. Lower panel: in the absence of serum (-FCS) all three WW domains of rNedd4 exclusively bound to the non-phosphorylated form of Cx43 (P0; arrows). (B) To strongly increase the phosphorylated forms of Cx43 before performing pull downs with GST-WW1, GST-WW2 and GST-WW3, confluent serum-starved (-FCS) WB-F344 cells were treated with EGF (+EGF). Bound Cx43 were then analyzed by SDS-PAGE and immunoblotting. The second lanes were loaded with cell lysates of EGF-treated WB-F344 cells as a size control for phosphorylated Cx43 molecules. Upper panel: incubation with anti-Cx43 antibody 71-0700 resulted in a strong signal for Cx43 in the WW2 and WW3 precipitates. Under these conditions, domain WW3 bound  10 times more Cx43 than domain WW2. Although binding of the WW1 domain to Cx43 was detectable in the immunoblot, the binding capacity of the WW1 domain was 5-10 times lower than that of domain WW2. Lower panel: with antibody SA226P, that specifically recognises the S279/S282 phosphorylated forms of Cx43, only a faint band in the WW2 precipitate was detectable, whereas no binding of S279/S282 phosphorylated Cx43 to the rNedd4 domains WW1 and WW3 was visible. P0 indicates the non-phosphorylated Cx43 molecules. P1 and P2 label putatively different phosphorylation forms of Cx43.
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) and Cx43 CT (
) against GST-WW2 concentration. The lines represent the best fit to the equation RU=RUmax/(1+KDapp/S). RU and RUmax are the resonance and the maximal resonance of bound GST-WW2, S is its free concentration, and KDapp is the apparent equilibrium dissociation constant. KDapp values (in µM) are listed as means ± s.e.m. in the table. NB, no binding. (B) GST-WW2 fusion protein (0.39 µM) was pre-incubated with increasing concentrations of non-biotinylated Cx43 CTphosph peptide, and the mixture was applied to a sensor chip with immobilized Cx43 CTphosph peptide at its surface. The steady-state signal B was expressed as B/Bmax, where Bmax is the steady-state signal at zero competing peptide. Binding of GST-WW2 to the immobilized Cx43 CTphosph decreased with increasing amounts of competing peptides.