IHEM   20887
INSTITUTO DE HISTOLOGIA Y EMBRIOLOGIA DE MENDOZA DR. MARIO H. BURGOS
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Production and characterization of a polyclonal antibody against 14-3-3ack49 to study 14-3-3 regulation
Autor/es:
FRONTINI LÓPEZ, Y. R.; BUSTOS, D. M.; MASONE, D.; UHART, M.
Lugar:
Buenos Aires
Reunión:
Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017
Institución organizadora:
Sociedad Argentina de Investigación Clínica (SAIC)
Resumen:
Post-translationalmodifications (PTMs) of proteins affect almost all cellularprocesses. Among them, acetylation of lysines (K) has emergedrecently as a reversible and dynamic PTM with important biologicalfunctions. The 14-3-3 proteins regulate the function and subcellularlocalization of thousands of proteins, through interaction withspecific phospho- serine and threonine residues. The acetylation of aspecific K (K49) which is part of the 14-3-3 binding pocket and isessential for its function, results in an inactive 14-3-3. To studyits regulation and the crosstalk mechanism betweenacetylation-phosphorylation, we produced an antibody to recognize theabove-mentioned AcK49 in 14-3-3. A chemically synthetized 15 aminoacid peptide, including the acetylated K49, and conserved in all14-3-3 paralogs, was coupled to Bovine Serum Albumin (BSA) ascarrier. This antigen was used to immunize rabbits in a pathogen-freeanimal facility (ISAL, UNL-CONICET). After 4 immunizations, blood wasdrawn from the animals, and the serum obtained by centrifugation. Thelater was tested by Dot- and Western blot against different 14-3-3constructions. It recognized all the acetylated peptide or complete14-3-3 forms (the antigen, an unconjugated synthetic peptide and arecombinant peptide coupled to Histone H3, all corresponding to thesame sequence in 14-3-3, and in vitro acetylated 14-3-3), but not thenon-acetylated recombinant 14-3-3. The serum did not recognize therecombinant peptide-H3 when blocked with the synthetic peptide. Asexpected, the preimmune serum did not recognize the antigen. This wasthe first step in a project to study 14-3-3 regulation byacetylation, which means regulation of regulators. We produced andcharacterized a polyclonal antibody that recognizes site-specificallythe acetylation of 14-3-3 on its K49, which at the best of ourknowledge is not commercially available.