Afters washes, cells were sequentially incubated with primary antibodies and with Cy2, Cy3 and Cy5 coupled anti rab bit or anti mouse secondary antibodies. DNA was stained using DAPI. Preparation were analyzed with Zeiss Laser Scanning confocal microscope LSM510. Images were acquired using 63X objective selleck chemical Abiraterone and Z series were projected onto a single view. Images were processed using Photoshop 8. 0. 1. Results FOP FGFR1 fusion protein interacts with CAP350 at the centrosome To better understand the role of the fusion protein at the centrosome, we looked for centrosomal interacting pro teins. Since FOP interacts with CAP350 we first deter mined that Inhibitors,Modulators,Libraries FOP FGFR1 and CAP350 colocalize at the centrosome in HeLa cells transiently transfected with FOP FGFR1.

Since FOP interacts with the C terminal domain of CAP350, we tested the ability of FOP FGFR1 to interact with this region of Inhibitors,Modulators,Libraries CAP350. Experi ments were done with lysates from Cos 1 cells transiently transfected with either CAP350 full length or CAP350 C terminal domain, and with either FOP FGFR1 or FGFR1. Immunoprecipitation Inhibitors,Modulators,Libraries with anti FGFR1 antibody and western blot analysis with anti CAP350 antibody showed that FOP FGFR1 interacts with CAP350 and that this implies the C terminal domain of CAP350. As expected, FGFR1 did not co immunoprecipitate with CAP350 confirming that the interaction is due to the FOP moiety of the fusion protein. The interaction is not dependent on FOP FGFR1 phosphorylation since the kinase defective FOP FGFR1 mutant protein could also interact with CAP350.

CAP350 Inhibitors,Modulators,Libraries depletion prevents FOP FGFR1 centrosomal localization CAP350 was depleted using two different oligonucle otides duplexes. Depletion effi ciency was demonstrated by both western blotting and immunofluorescence. CAP350 depletion abolished the association of FOP FGFR1 with centrosomes in interphase and M phase cells. FOP FGFR1 recruits the p85 subunit of PI3K at the centrosome during interphase We studied the localization of p85 PI3K subunit in FOP FGFR1 expressing cells. We found that p85 has a diffuse localization in the cytoplasm of wild type Ba/F3 cells, whereas it is efficiently recruited at the centrosome by FOP Inhibitors,Modulators,Libraries FGFR1 in interphase, and weakly during mitosis. This recruitment requires phos phorylated FOP FGFR1 as it did not occur in cells express ing the kinase defective FOP FGFR1 K259A mutant although this mutant protein also localized to the centrosome.

selleck screening library To test the specificity of the inter action of p85 with FOP FGFR1, we wondered if other fusion kinases also recruit p85. Two other fusion kinases tested are not located at the cen trosome. CEP110 FGFR1 was the only one to provide the pYXXM motif at the centrosome. However, we could not observe p85 recruitment at the centrosome in Ba/F3 cells expressing CEP110 FGFR1. This sug gests that the pYXXM motif is not sufficient to recruit p85 at the centrosome.