Peroxidaseconjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and a luminescence system (PerkinElmer, Waltham, MA, USA). For the protein loading manage, membranes have been incubated with an antiactin Santa Cruz Biotechnology (Santa Cruz, CA, USA) antibody. Protein expression was quantified employing the BioRad Quantity One 1D Analysis software (BioRad Laboratories, Inc., Hercules, CA, USA). The levels of phosphorylated proteins: phosphoS6 Ser235236, phosphoAKT Ser 473, and pERKS were normalized by the levels of their corresponding total protein (total, S6, and AKT), all others have been normalized by loading handle (actin). The levels of expression of phosphorylated proteins and their corresponding total protein were evaluated within the similar gel, additionally, the antibodies utilized for the total proteins recognize all forms of the phosphorylated proteins. four.8. Statistical Evaluation Statistical evaluation was performed with SPSS version 21.00 (SPSS Inc). The expression of phosphoAKT Ser473 is expressed as mean typical deviation. An independent sample Student’s t test was employed to evaluate feasible associations involving phosphoAKT Ser 473 expression and Flusilazole manufacturer clinicopathological and molecular attributes to evaluate protein expression (analyzed by western blot) between groups. A Pearson Correlation was used to evaluate the correlation involving phosphoAKT Ser473, phosphomTOR Ser2448, and phosphoS6 Ser235236 expression. A Chisquare test wasInt. J. Mol. Sci. 2018, 19,13 ofused to evaluate probable associations among phosphoAKT Ser 473 nuclear expression and clinicopathological and molecular characteristics. Benefits had been viewed as statistically substantial at p 0.05.Supplementary Components: Supplementary materials could be found at http:www.mdpi.com142200671951448 s1. Author Contributions: C.T. and P.S. conceived and designed the experiments; C.T., A.P., R.B., A.G., and D.R. performed the experiments; C.T., P.S., M.M., C.E., and L.B.F. analyzed the information; M.S.S., C.E., and E.R. performed the histological revision from the instances; C.T. and P.S. wrote the paper; P.S. and M.S.S. revised the paper. Acknowledgments: This study was supported by FCT (“Portuguese Foundation for Science and Technology”) by means of PhD grants to Catarina Tavares (SFRHBD878872012), Ana Pestana (SFRHBD1106172015), and Rui Batista (SFRHBD1113212015) and by a CNPq PhD grant (“National Counsel of Technological and Scientific Development”, Brazil), Science without having Borders, Method n 23732220129 for Luciana Ferreira. Miguel Melo received a grant from Genzyme for the analysis project “Molecular biomarkers of prognosis and response to therapy in differentiated thyroid carcinomas”. Additional funding was obtained from FEDERFundo Europeu de Desenvolvimento Regional funds by way of the COMPETE 2020Operational PEG4 linker Antibody-drug Conjugate/ADC Related Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCTFunda o para a Ci cia e a TecnologiaMinist io da Ci cia, Tecnologia e Inova o within the framework on the project “Institute for Investigation and Innovation in Well being Sciences” (POCI010145FEDER007274), and by the project “Advancing cancer investigation: from fundamental acknowledgement to application”; NORTE010145FEDER000029; “Projetos Estruturados de I D I, funded by Norte 2020Programa Operacional Regional do Norte. This operate was also financed by Sociedade Portuguesa de Endocrinologia Diabetes e Metabolismo via a grant “Prof. E. Limbert Sociedade Portuguesa de Endocrinologia Diabetes e MetabolismoSanofiGenzyme i.