SUMOylation of N-Myc in neuroblastoma cells.

<p>(<b>A</b>) Immunoblot analysis in denaturing conditions of different neuroblastoma cell lines after 6 h treatment with 10 µM MG132. (*): non-specific band. (<b>B</b>) Immunoprecipitation (IP) with anti-N-Myc antibodies in denaturing conditions and analysis by immunoblotting with the indicated antibodies. (<b>C</b>) SHSY5Y cells were infected with pQCXIP retroviral vectors expressing Flag-HA tagged WT or K349R N-Myc proteins and expression of selected mRNAs was measured by RT-PCR (after normalization to the housekeeper RPPO). The histogram represents the mean and s.d. of three independent experiments. The total levels of the indicated proteins were assessed by immunoblot. (<b>D-F</b>) SK-N-BE(2) cells were infected with pQCXIN empty vector, Flag-HA-N-Myc WT or K349R (mouse cDNA) and superinfected with pGIPZ-PURO shN-Myc or control shRNA. (<b>D</b>) Immunoblot analysis of the level of endogenous and Flag-HA tagged exogenous N-Myc protein in mock treated cells and cells treated with MG132 10 µM. (<b>E</b>) FACS analysis of BrdU incorporation (% of positive cells); the histogram represents the mean and s.d. of three independent experiments. The numbering of the samples corresponds to that in (<b>D</b>). In neither of the quantitative assays used (panels C, E) was a statistically significant difference observed between the WT and K349R forms of N-Myc. (<b>F</b>) Colony Assay. Cells were plated in 6-well plates, incubated for 7 days and stained with Crystal violet. Numbers in each plate indicate relative cell densities, as assessed by absorbance at 595 nm following solubilization of the dye with acetic acid.