These sub strates could either be signaling molecules phosphor ylated at the centrosome or intrinsic centrosomal proteins. In this report, we have characterized the interaction of FOP FGFR1 with CAP350, and shown that the localiza tion of the tyrosine kinase at the centrosome induces the recruitment in selleck chem Perifosine this specific area of two enzymatic sub strates, PI3K and PLC?1. Methods Plasmids, cells and reagents Ba/F3 cells hematopoietic cells were grown as described. HeLa and Cos 1 cells were grown in DMEM with 10% fetal calf serum. Myc tagged FOP FGFR1 and myc tagged FOP FGFR1 kinase defective and corre sponding clones of stably transfected Ba/F3 cells have been previously described. Ba/F3 stably expressing BCR ABL or the EPO receptor are a gift from P. Dubreuil.

GFP tagged FOP FGFR1 was obtained by inser tion in pEGFP vector conferring GFP tag at the N termi nus. The quickchange site directed mutagenesis Inhibitors,Modulators,Libraries kit from Stratagene was used to introduce point mutations changing tyrosine to phenylalanine at position 511 and 475 of FOP FGFR1. Each construct was verified by sequencing. Myc tagged CAP350 and myc tagged CAP350 C terminal Inhibitors,Modulators,Libraries domain constructs were obtained by subcloning in pCS2 with a myc tag. The plasmid which direct the GST SH2 N terminal p85has been described. Transient and sta ble transfections were done as described. Antibodies We used monoclonal anti myc, polyclonal anti FGFR1, and polyclonal anti PLC?1, from Santa Cruz Biotechology, polyclo nal anti p85 from Upstate Biotechnology, monoclonal anti p85 and anti GFP from Abcam, polyclonal anti pYXXM and polyclonal Inhibitors,Modulators,Libraries anti pPLC?1 from Cell Signaling, anti ? tubulin either mon oclonal or polyclonal from Sigma Aldrich.

Polyclonal anti CAP350 antibody was obtained by immunizing rabbits against the AA 1875 2055 domain of CAP350. Cell lysis, Inhibitors,Modulators,Libraries immunoprecipitations, Inhibitors,Modulators,Libraries western blot and GST pull down Ba/F3 cells were starved over 7 h in RPMI containing 0. 5% FCS. For immunoprecipitation assays, cells were lysed in NP40 1% lysis buffer. Protein extracts were incubated with antibodies and subsequently with either protein A or G agarose. Bound proteins or total protein extract were separated as described or using NuPAGE 4 12% Novex Bis Tris gels according to the manufacturers instructions for CAP350 experiments. For GST pull down experiments, 10 g of bacterially product GST p85 or GST alone were used as previously described. Cell survival assays 1. 106 Ba/F3 cells were washed three times and grown in triplicate in the absence of IL3. The number of viable cells was measured by trypan blue exclusion. For PI3K inhibi tion experiments, 10 M LY294002 was used. Immunofluorescence and confocal analysis HeLa cells were grown on coverslips and starved Ba/F3 cells were centrifugated Tofacitinib JAK3 to adhere to the coverslips.