Fig. 4. p120 phosphorylation at Y228 during adherens junction formation. (A) Characterization of a p120 knockdown and add-back model system. Endogenous p120 levels were stably knocked down with retrovirally transduced siRNA specific for human p120 (pRS.h) in parental A431 cells, and clonally derived pRS.h-expressing A431 cell lines were subsequently infected with retroviral vector alone or murine p120 constructs WT-mp120-3A or mp120-3A/7F (which contains phenylalanine mutations at the seven known p120 Y phosphorylation sites). Murine p120 is unaffected by the human p120 siRNA because of mismatches at the nucleotide level. RIPA lysates were probed by western blotting. Human and mouse p120 together were detected by western blotting with mAb pp120 (Total p120). Tubulin was simultaneously detected as a loading control. Murine p120 (mp120-3A) was selectively detected with the murine-specific mAb 8D11. E-cadherin expression was detected with HECD-1. (B) Morphologic effects of WT- and 7F-p120 expression in p120 knockdown cells. The cells lines in A were trypsinized, plated overnight and photographed on a phase contrast microscope (10x magnification). No differences were detected between cells rescued with wild type (mp120-3A) and mutant (mp120-3A/7F) p120. (C) Effects of junction formation on p120 Y228 phosphorylation. p120 Y228 phosphorylation was monitored by western blotting of cell lysates after calcium switch (see Materials and Methods). Total p120 (p120) and Y228 phosphorylated p120 (pY228) were quantitated in the presence of calcium (NS=no switch), or at the indicated times after calcium add-back. (D) Effects of EGFR inhibition. The experiment in C was repeated in the presence of the EGFR inhibitor AG1478 (300 nM). (E) A431 pRS.h (p120 knockdown) cells expressing mp120-3A or mp120-3A/7F were subjected to calcium switch assay as in C, and then examined by immunofluorescence with p120-specific antibody pAb F1SH at the indicated times after add-back of calcium (63x magnification).