Figure 3

V5 immunoprecipitation experiments and western blots. (A) Kelly cells were transfected with PTPRD cDNA or empty vector (pcDNA3.1-V5). V5 Immunoprecipitates were subjected to western blot with an AURKA antibody in order to confirm in vivo association of AURKA and PTPRD. (B) Western blot of the same immunoprecipitates with a V5 antibody demonstrates the pull down of V5 PTPRD. (C) Kelly cells were transfected with PTPRD cDNA, empty vector or PTPRD mutant (Q1481X). Post pan-phosphotyrosine immunoprecipitation (pY) and western blotting with AURKA reveals the phosphorylation status of AURKA in these transfectants. Immunoprecipitation of the V5 tag and subsequent western blotting with V5 confirms upregulation of V5-PTPRD (D) Ectopic up-regulation of PTPRD results in destabilization of AURKA. Kelly cells were transfected with PTPRD cDNA, empty vector or the phosphatase dead PTPRD mutant Q14811. Lysates were harvested 72 hours post transfection and treated at 0, 1, 2, 3, 4 and 6 hours with cycloheximide and then subjected to western blot analysis with an AURKA antibody. Alpha tubulin was used as a loading control. (E) The graph shows quantitative densitometry of the protein expression of AURKA (n = 2) following normalization with the endogenous control. (F) Ectopic up-regulation of PTPRD results in a decrease in protein levels of AURKA. and MYCN. Increasing concentrations of PTPRD cDNA were transfected into Kelly cells. Lysates were harvested 72 hours post transfection and subjected to western blot analysis with V5, AURKA and MYCN antibodies. Alpha tubulin was used as a loading control