Supplement Figure 5

1. (XB130) (XB130) Supplement Figure 5 A B C

2. Supplement Figure 5. Inhibition of RET kinase with ZD647 in TPC1 cells containing the RET/PTC rearrangement leads to decrease in XB130 tyrosine phosphorylation and reduced binding to p85. A) TPC1 cells expressing endogenous XB130 and RET/PTC were treated with DMSO or 1 μM of RET kinase inhibitor, ZD6474, for 48 h. Interaction between endogenous XB130 and p85 were examined by immunoprecipitation using anti-XB130 antibody followed by western blotting with antibodies against XB130 and p85. B) Cell lysates from TPC1 cells treated with or without ZD6474 were incubated with glutathione-Sepharose-imobilized GST-p85 N-terminal or C-terminal SH2 domain of p85. The pull down samples and total cell lysates were immunoblotted using anti-XB130 antibody. The minus lanes in panels A and B are no protein lysate as negative controls.C) Inhibition of RET/PTC kinase activity with the ZD 6474 leads to decrease in XB130 phosphorylation and its binding to p85. TPC1 cells were transfected with myc-tagged XB130, 48 h following transfection cells were treated with ZD6474 for different periods. Collected cell lysates were immunoprecipitated using anti-myc antibody and immunoblotted using anti-pY and anti-p85 antibodies. Total cell lysates were analyzed for the levels of exogenous XB130 with anti-Myc antibody and RET phosphorylation using anti-pY905 antibody.