Epoxomicin

TDP-43 degradation was analyzed in N2a cells plated in 6-well plates (30,000 cells/well density) at 0 min, 30 min, 1 h, 2 h, 3 h, 15 h, and 24 h after transfection. In a set of experiments, the cells were pre-treated with 100 nM epoxomicin (Abcam, Cambridge, UK), a potent proteasome inhibitor [53 (link)], or with 400 nM bafilomycin (Abcam, Cambridge, UK), which inhibits autophagic flux by preventing the acidification of endosomes and lysosomes [54 (link)]. Then, the cells were washed with PBS, fixed in 2% (w/v) buffered paraformaldehyde for 10 min at room temperature (20 °C), and permeabilized with a 0.5% (v/v) Triton X-100 solution for 5 min. Then, the cells were incubated for 60 min at 37 °C with 1:500 mouse monoclonal anti-TDP-43 antibodies (Novus Biologicals, Littleton, CO, USA) or with 1:500 rabbit polyclonal anti-murine TDP-43 antibodies (LSBio, Seattle, WA, USA), which only recognize the murine protein, and for 90 min with 1:1000 diluted Alexa Fluor 488-conjugated anti-mouse or 594-conjugated anti-rabbit secondary antibodies, respectively, and then analyzed by confocal microscopy, as described above.