Skip to main content
. 2015 Oct 6;290(47):28200–28213. doi: 10.1074/jbc.M115.655597

FIGURE 2.

FIGURE 2.

Expression of NRBE3 resulted in an active proteolysis of RB. A, U2OS cells were transfected with increasing amounts of FLAG or FLAG-NRBE3 plasmid and the same dose of GFP plasmid. Western blotting was performed with proteins from cell lysates. The upper part of the blot was probed with anti-FLAG, and the lower part was probed with anti-RB antibody. GFP was evaluated as a transfection efficiency control, and β-actin was evaluated as a loading control. LE, long exposure bands; SE, short exposure bands. B, U2OS cells were transfected with FLAG-NRBE3 expression plasmid. Cells were fixed 24 h post-transfection, and double immunofluorescence staining was performed with monoclonal anti-RB antibody and polyclonal anti-NRBE3 antibody. Immunocomplexes were probed with TRITC-conjugated goat anti-rabbit IgG and FITC-conjugated goat anti-mouse IgG. Nuclei were stained with DAPI. The image was taken under confocal microscopy. Scale bars represent 50 μm. C, HCT116 cells were transfected with NRBE3-specific siRNAs or a control siRNA, respectively. Western blotting was performed with proteins from cell lysates. The upper part of the blot was probed with anti-NRBE3 antibody, and the lower part was probed with anti-RB antibody. β-Actin was evaluated as a loading control. -Fold induction of the relative protein levels of RB is summarized from three independent experiments. Error bars represent S.E. *, p < 0.05 versus untreated cells (right panel). D, U2OS cells were transfected with either FLAG-NRBE3 or FLAG vector plasmid. Cells were treated with 10 μg/ml cycloheximide at 16 h post-transfection. Cells were harvested at the indicated time points, and cell lysates were prepared. Proteins from cell lysates were subjected to Western blotting with anti-FLAG and anti-RB antibodies as described in A (upper panel). GFP and β-actin were evaluated as transfection efficiency and loading controls, respectively. Relative RB levels were plotted with the integrated optical density of the RB bands on the Western blot (lower panel). E, U2OS cells were transfected with NRBE3-specific siRNAs or a control siRNA, respectively. Cells were treated with 10 μg/ml cycloheximide at 48 h post-transfection. Cells were harvested at the indicated time points, and cell lysates were prepared for Western blotting as described in A (upper panel). Relative RB levels were plotted with the integrated optical density of the RB bands on the Western blot (lower panel).