PMC2360227_01
Upper panel. FISH analysis. A probe mix for PDGFRB (BAC clone RP11-368O19) (in green)/a locus specific in 5p (in red), PDGFRA (BAC clone RP11-231C18) (in green)/Cep4 (in red) (Vysis) and c-Kit (BAC clone RP11-586A2 (in green)/Cep4 (in red) (Vysis), respectively, was employed to detect gene status for the receptors. A condition of high polisomy was detected for PDGFRB, PDGFRA and c-Kit. Lower panel. Immunoprecipitations/Western blot analysis. Equal amounts of total protein extracts (1 mg) were immunoprecipitated using the specified antibodies (PDGFRB sc 432, Santa Cruz Biotechnology; PDGFRA 07-276, Upstate, Charlottesville, VA, USA and KIT Ab-3, clone K45, Neomarkers, Fremont, CA, USA) subsequently blotted to a membrane and incubated first with anti p-Tyr antibody (clone 4G, Upstate) and then with antibodies for the specified receptors. As positive controls, for PDGFRA and PDGFRB, an NIH3T3 cell line was employed, whereas for KIT a Delta559 cell line expressing a constitutively activated KIT receptor was used.
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PMC2360227_01_6603542f1.jpg
fig1
The primary lung tumour was investigated in addition to FISH analysis, by IHC, IP/WB and DNA sequencing (after the informed consent of the patient) (Figure 1).