Resumen de Intracellular distribution of the ?Np73 protein isoform in medulloblastoma cells: a study with newly generated rabbit polyclonal antibodies

Renata Veselska, Jakub Neradil, Marta Nekulova, Lucia Dobrucka, Borivoj Vojtesek, Jaroslav Sterba, Karel Zitterbart

• The p73 protein is a member of the p53 family of transcription factors that has two N-terminal isoforms: the TAp73 isoform is reported to have a tumor suppressor function, whereas the ?Np73 isoform likely has oncogenic potential. The expression of these isoforms and the differences in their intracellular distribution have been described in many cancer types; however, little is known about the p73 isoforms in brain tumors. Our study is focused on the intracellular localization of ?Np73 in medulloblastoma cell lines. Due to a lack of suitable anti-?Np73 antibodies, we developed two new rabbit polyclonal antibodies, ?Np73-26 and ?Np73-27, with sufficient specificity, as demonstrated by immunodetection methods using transiently transfected cell lines. Both of these new antibodies were subsequently used for analysis of the ?Np73 distribution in medulloblastoma cells using immunofluorescence, immunoblotting and immunogold labeling for transmission electron microscopy. We found a nuclear localization of the ?Np73 isoform in all of the medulloblastoma cell lines included in this study. Furthermore, a non-random accumulation of the ?Np73 isoform near the cell nuclei was observable in all of these cell lines. By double-labeling with ?Np73 and golgin-97, we showed the co-localization of the ?Np73 isoform with the Golgi apparatus. Nevertheless, further detailed analyses of possible interactions of ?Np73 with the proteins accumulated in the Golgi apparatus should be performed to explain the dynamics of ?Np73 outside the cell nucleus.