Effect of internal deletions within the N-terminal region of Nurr1 in its degradation.

<p>HeLa cells were transiently transfected with full-length Nurr1, Nurr1 Δ163–187, Δ163–217 and Δ163–249 as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (A). Protein loading control was assessed by immunobloting with anti-tubulin antibodies (A). (B) Graph shows the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. (C) Cells were also analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for Nurr-1 subcellular localization.</p

Effect of treatment of cells with Leptomycin B on the degradation of Nurr1.

<p>HeLa cells were transiently transfected with full-length Nurr1, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) or Leptomycin B for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies Protein loading control was assessed by immunobloting with anti-tubulin antibodies. Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments.</p

Effect of the deletions from the N-terminal of Nurr1 on its degradation.

<p>HeLa cells were transiently transfected with full-length Nurr1, Δ1–262, Δ1–161 and Δ1–96 (A) or Nurr1 Δ1–80, Δ1–63, Δ1–43, and Δ1–31 (C) as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (A and C). Protein loading control was assessed by immunobloting with anti-tubulin antibodies. (B and D) Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. (E) Cells were also analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for Nurr-1 subcellular localization of the different Nurr1 constructs as indicated.</p

Degradation of endogenous Nurr1 in PC12 cells and ectopically expressed Nurr1 in HeLa cells.

<p>(A) PC12 cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies. (B, D and F) HeLa cells were transiently transfected with full-length Nurr1, N-terminal flag-tagged Nurr1 or Nurr1 1–337 as indicated, after transfection cells were treated with CHX in the absence or in the presence of Lactacystin (Lacta) for the times indicated and cell extracts analyzed by immunoblot with anti-Nurr1 antibodies (B and D). Protein loading control was assessed by immunobloting with anti-tubulin antibodies. (C and E) Graphs show the quantification of immunoblots, and results are expressed as means ± s. e. m. from three different experiments of the indicated Nurr1 protein constructs. F, transfected cells were analyzed by indirect immunofluorescence with anti-Nurr1 antibodies (red channel), counterstained for nuclei with DAPI (blue channel) and imaging by confocal microscopy for subcellular localization of Nurr1.</p

Ubquitylation of Nurr1 and Nurr1 Δ1–31.

<p>HeLa cells were co-transfected with Nurr1 full length or Δ1–31 and HA-ubiquitin and either untreated or treated with lactacystin (Lacta) as indicated, cell extracts were immunoprecipatated with anti-HA antibodies, analyzed by SDS-PAGE and immunoblotted with anti-Nurr1 antibodies (A). (B) Direct immunoblot with anti-Nurr1 antibodies of 1/10 of the amount of total cell extracts (shorter exposure than the upper panel) used for immunoprecipitation experiments shown in the upper panel. Protein loading control was assessed by immunobloting with anti-tubulin antibodies.</p