Thogenfree housing conditions. To activate the transactivating function in the rtTA protein, mice were fed with rodent chow containing 200 mg/kg Dox (Dox diet program, Bio-Serv). Animal research and care have been authorized by the institutional animal care and use committee on the University of South Florida and followed institutional and national guidelines. Reverse transcription CR analysis of SHP2E76K messenger RNA expression Tissue samples had been snap frozen in liquid nitrogen. RNA was extracted using Trizol reagent (Life Technologies). Samples had been treated with DNase I (Life Technologies) to prevent DNA contamination and reverse transcription CR (RT CR) was performed using the SuperScript One-Step RT CR Platinum Taq system (Life Technologies) with all the following primers: SHP2F1: 5-GGTTGGACAAGGGAATACGG-3 and SHP2R2: 5-AGGGCTCTGATCTCCACTCG-3. The protocol for a 50 l RT CR reaction was as follows: 30 min complementary DNA synthesis at 55 , four min denaturation at 94 then 35 cycles of 94 for 30 s, 57 for 30 s, then 72 for 30 s with a final extension step of 72 for 4 min, which yields a 462 bp fragment. Histological and immunohistochemical examination Right after euthanasia, the mouse lungs had been flushed twice with 10 ml phosphatebuffered saline and insufflated with ten buffered formalin. Right after fixation overnight in ten buffered formalin remedy at room temperature, paraffin blocks have been prepared by regular process by the Histology Service with the Tissue Core from the Moffitt Cancer Center. Sections (four m thick) had been stained with hematoxylin and eosin (H E) for histological examination. For immunohistochemical evaluation of pErk1/2, slides had been stained employing a Ventana Discovery XT automated technique (Ventana Health-related Systems, Tucson, AZ). Slides have been deparaffinized with EZ Prep option (Ventana). Heat-induced antigen retrieval strategy was utilized in Cell Conditioning 1 (Ventana).Hematoxylin Epigenetic Reader Domain A rabbit anti-pErk1/2 (#4376, Cell Signaling, Danvers, MA) was utilized at a 1:200 dilution in PSS diluent (Ventana) and incubated for 32 min. Anti-rabbit secondary antibody (Ventana) was used for 20 min. The detection method utilized was the Ventana OmniMap kit and slides had been counterstained with hematoxylin. Immunoblotting, immunoprecipitation, kinase assay and mass spectrometry Antibodies to SHP2, Erk1/2, phospho-Erk1/2 (pErk1/2), Gab1, Akt, c-Myc and -actin had been obtained from Santa Cruz Biotechnology (Santa Cruz, CA).Flag (rabbit), pGab1 (Y627), phospho-Akt (pAkt) and phospho-Src (pSrc) antibodies have been from Cell Signaling Technologies.Nonactin custom synthesis Anti-Src antibody was from Calbiochem (Billerica, MA) and M2 Flag antibody was from Sigma (St Louis, MO).PMID:23795974 Antibodies to MDM2 (clone 2A9) and MDMX (clone 8C6) have been as described (38,39). The anti-p53 antibody was from IMGENEX (San Diego, CA). Frozen tissues had been crushed and lysed with lysis buffer (50 mM Tris Cl, pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 25 mM NaF, five mM Na4P2O7, 1 mM dithiothreitol, 1 mM Na3VO4, 100 g/ml phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, two g/ml aprotinin and 1 Triton X-100). Equal amounts of proteins from cleared tissue lysate supernatants have been separated by ten sodium dodecyl sulfate olyacrylamide gels and transferred to nitrocellulose filters for immunoblotting. Flag-tagged SHP2 was immunoprecipitated from cleared tissue lysate supernatants by using the anti-Flag M2 antibody and Protein-G agarose. Immunoblotting was performed as described previously (15,29). Ce.