FIGURE 5.

Immunofluorescence staining and Western blotting analyses of Neuro2A cells treated with siRNA oligonucleotides and MG132. Neuro2A cells were transfected with the scRNA oligonucleotide (upper panels) or siRNA-1 oligonucleotide (lower panels), treated with MG132 overnight, and then immunofluorescence co-stained with anti-TDP-43 (green) and anti-ubiquitin (Ubi; red). The nuclei were stained with DAPI. Note the much higher ubiquitin immunoreactivity in cells without TDP-43 due to knockdown by siRNA-1, as exemplified by the arrow in cells in the bottom panels. The arrowheads in the far right panel of the bottom panels point to those cells maintaining TDP-43 and with low ubiquitin immunoreactivity. The quantitative comparison of the average intensities of anti-ubiquitin/cell of Neuro2A cells treated with scRNA, siRNA-1, (scRNA+MG132), and (siRNA-1+MG132), respectively, is shown in the lower left histogram. *, p value < 0.05; **, p value < 0.01. B, Western blotting analysis of total proteins extracted in urea buffer from Neuro2A cells transfected with scRNA oligonucleotide (lanes 1 and 3) or siRNA-1 oligonucleotide (lanes 2 and 4) with or without MG132 treatment for overnight (lanes 3 and 4), with the use of anti-ubiquitin (left panel), anti-p62, anti-TDP-43, and anti-tubulin (right panels) as the probes. Note the higher ubiquitin (Ub) immunoreactivity (left panel) and higher amounts of p62 proteins (right panel and the histogram) in samples treated with siRNA-1 and/or MG132. The histogram shows quantitative data. *, p value < 0.05; ***, p value < 0.001. arb. unit; arbitrary unit.