Ined in precise pathogenfree housing situations. To activate the transactivating function in the rtTA protein, mice were fed with rodent chow containing 200 mg/kg Dox (Dox diet, Bio-Serv). Animal research and care were approved by the institutional animal care and use committee in the University of South Florida and followed institutional and national recommendations. Reverse NK1 Antagonist Species transcription CR analysis of SHP2E76K messenger RNA expression Tissue samples were snap frozen in liquid nitrogen. RNA was extracted working with Trizol reagent (Life Technologies). Samples have been treated with DNase I (Life Technologies) to avoid DNA contamination and reverse transcription CR (RT CR) was performed making use of the SuperScript One-Step RT CR Platinum Taq program (Life Technologies) using the following primers: SHP2F1: 5-GGTTGGACAAGGGAATACGG-3 and SHP2R2: 5-AGGGCTCTGATCTCCACTCG-3. The protocol to get a 50 l RT CR reaction was as follows: 30 min complementary DNA synthesis at 55 , 4 min denaturation at 94 then 35 cycles of 94 for 30 s, 57 for 30 s, then 72 for 30 s using a final extension step of 72 for 4 min, which yields a 462 bp fragment. Histological and immunohistochemical examination Soon after euthanasia, the mouse lungs were flushed twice with 10 ml phosphateMEK Inhibitor Storage & Stability buffered saline and insufflated with 10 buffered formalin. Soon after fixation overnight in 10 buffered formalin resolution at room temperature, paraffin blocks had been ready by normal process by the Histology Service of the Tissue Core in 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 were stained using a Ventana Discovery XT automated program (Ventana Health-related Systems, Tucson, AZ). Slides were deparaffinized with EZ Prep remedy (Ventana). Heat-induced antigen retrieval system was made use of in Cell Conditioning 1 (Ventana). A rabbit anti-pErk1/2 (#4376, Cell Signaling, Danvers, MA) was applied at a 1:200 dilution in PSS diluent (Ventana) and incubated for 32 min. Anti-rabbit secondary antibody (Ventana) was employed for 20 min. The detection method utilized was the Ventana OmniMap kit and slides were 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 were from Cell Signaling Technology. Anti-Src antibody was from Calbiochem (Billerica, MA) and M2 Flag antibody was from Sigma (St Louis, MO). Antibodies to MDM2 (clone 2A9) and MDMX (clone 8C6) had 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, 5 mM Na4P2O7, 1 mM dithiothreitol, 1 mM Na3VO4, 100 g/ml phenylmethylsulfonyl fluoride, 2 g/ml leupeptin, 2 g/ml aprotinin and 1 Triton X-100). Equal amounts of proteins from cleared tissue lysate supernatants were 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 pre.
