Nuclear localization of GNL3L modulates Rb-E2F1 pathway.

<p>Ectopic expression of GNL3L^ΔNES leads to hyperphosphorylation of Retinoblastoma protein (Rb) at serine 780. HEK293 <b>(A)</b> and MCF-7 <b>(B)</b> cells were transfected with wild type or variants of GNL3L. Anti-phospho Rb (S780) antibody and anti-Rb antibodies were used to determine the status of Rb phosphorylation and total Rb protein levels, respectively, by western blot analysis. Beta actin served as loading control. (<b>C</b>) GNL3L^ΔNES expression leads to increased association between cyclin D1 and cdk4. HEK293 cell lysates containing wild type or variants of GNL3L were subjected to co-immunoprecipitation with anti-cyclin D1 antibody followed by western blot using anti-cdk4 antibody. Expression levels of the indicated variants of GNL3L were normalized to that of GNL3L^WT and used as the reference for these analyses. <b>(D)</b> GNL3L^ΔNES displays Rb dependency in the upregulation of E2F1, cyclins A2 and E1 levels. The Rb null cell line, Hep3B was transfected with wild type or variants GNL3L, alone or in combination with Rb expression plasmid. RT-qPCR was performed for the respective genes using specific primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0135845#pone.0135845.s004" target="_blank">S1 Table</a>).