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. 2003 Oct;23(20):7391–7402. doi: 10.1128/MCB.23.20.7391-7402.2003

FIG. 4.

FIG. 4.

Phosphorylation of plakoglobin by Src decreases its interaction with α-catenin and E-cadherin and increases plakoglobin-desmoplakin association. (A) Five picomoles of GST or GST-plakoglobin fusion proteins was phosphorylated with 0.5 U of pp60c-src. Pull-down assays were then performed by incubating the GST proteins with 100 μg of total cell extracts from SW480. The amount of associated proteins was determined by using specific MAbs. (B) Control and Src-phosphorylated plakoglobin (0.35 or 0.7 pmol) were incubated with 1.2 pmol of either GST-cytoE-cadh or GST in a final volume of 200 μl. Plakoglobin (0.03 pmol) was included as a reference (St). The numbers below the lanes indicate the amount of bound protein. (C) GST or GST-plakoglobin fusion proteins (control or phosphorylated by Src) (1.8 pmol) were incubated with 2.4 pmol of α-catenin in a final volume of 200 μl. The amount of bound α-catenin was determined with a specific MAb. (D) Modulation of plakoglobin-α-catenin association by E-cadherin. GST or GST-plakoglobin (0.8 pmol) was incubated with 1.4 pmol of α-catenin. When indicated, binding assays were supplemented with 20 pmol of cytoE-cadh. The amount of associated α-catenin was determined by using a specific MAb. (E) Modulation of plakoglobin-E-cadherin association by α-catenin. GST or GST-plakoglobin (0.8 pmol) was incubated with 1.4 pmol of cytoE-cadh. When indicated, binding assays were supplemented with 20 pmol of α-catenin. The amount of associated cytoE-cadh was determined by using a specific MAb. (F) GST or GST-plakoglobin fusion proteins (5 pmol) were phosphorylated with Src, and pull-down assays were then performed by incubating the GST proteins with 100 or 200 μg of total cell extracts from SW480. The amount of associated desmoplakin was determined by using a specific MAb. (G) GST or GST-plakoglobin (5 pmol) was incubated with 200 μg of total cell extracts from SW480 with the presence of 10 pmol of α-catenin or cytoE-cadh when indicated. The amount of associated desmoplakin was determined by using a specific MAb. WB, Western blotting; +, present; −, absent.