FIGURE 2.

p120ctn is necessary for PS1-cadherin interaction. A, HEK293 cell cultures depleted of p120ctn via shRNA (see “Materials and Methods”) were transiently transfected with zero (–), 0.1 (+), or 0.3 (++) μg of N-cadherin cDNA. A WT culture was used as a control. Extract from each culture was prepared in Nonidet P-40 and then immunoprecipitated with either anti-PS1/CTF (PS1 IP) or anti-N-cadherin (N-cad IP) antibodies. Obtained IPs were probed on WBs with antibodies against antigens indicated at the right of the figure. Note that PS1/CTF levels were not affected by the knockdown of p120ctn or the expression of exogenous N-cadherin. Reintroduction of N-cadherin did not restore the amounts of N-cadherin-PS1 complex in p120ctn knockdown cells. B, control or p120ctn-depleted (p120ctn KD) HEK293 cell cultures were transduced with pMX, WTPS1, or PS1/D257A mutant, and N-cadherin was reintroduced into p120ctn KD cultures as described above. Extracts prepared as described above were immunoprecipitated with anti-N-cadherin antibodies (N-cad IP), and obtained IPs were probed on WBs with antibodies against antigens indicated at the right of the figure. Input is shown in the left panels. Note that knockdown of p120ctn decreases the interaction between uncleaved PS1 and N-cadherin. The double signal of the N-cadherin in the N-cadherin-transfected cultures is due to the presence of unprocessed (immature) protein. Unidentified (artifactual) bands are indicated by an asterisk. C, p120ctn-deficient colon carcinoma SW48 cells were transduced with pMX vector (PMX), E-cadherin (E-cad), or p120ctn. Extracts from the obtained cultures were immunoprecipitated as described above with antibodies against PS1/CTF (IP α PS1/CTF), and IPs were probed on WBs with antibodies against antigens indicated at the right of the figure. Input is shown in the left panels. Results show that p120ctn is needed for E-cadherin/PS1 interaction.