Figure 3

The TDP-43 C-terminal fragment, GFP-TDP _220-414 , is preferentially degraded through the ubiquitin-proteasome pathway. To monitor GFP-TDP_220-414 degradation, M17D3 were grown in doxycycline-free media for 5 days to induce GFP-TDP_220-414 expression. Its expression was then inhibited by doxycycline and cells were treated with the proteasome inhibitor, MG-132, the lysosome inhibitors, chloroquine (CQ) or NH₄Cl, or the autophagy inhibitor, 3-MA. Control cultures were maintained in doxycycline-free media to sustain GFP-TDP_220-414 expression. On day 6, cells were harvested for Western blot analysis and confocal microscopy. (A) Blots were probed using an anti-cTDP-43, anti-pTDP-43 and anti-GAPDH antibodies. The arrow indicates GFP-TDP_220-414, the asterisk (*) endogenous TDP-43, and the dashed arrows cleavage products, likely resulting from GFP-TDP_220-414 truncation. Dox=doxycycline, HMW = high molecular weight, NS=non-specific. (B) GFP fluorescence in cells after 6 days of GFP-TDP_220-414 induction in doxycycline-free media (CTRL) was compared to that in cells treated with doxycycline during the last 24 h of the 6-day experiment (Dox) or to cells co-treated with doxycycline and MG-132 (Dox+MG-132), chloroquine (Dox+CQ), or 3-MA (Dox+3-MA). Scale bars, 20 μM (10 μM in inset). (C) To determine if the GFP-tag is responsible for targeting GFP-TDP_220-414 to the proteasome for degradation, M17D cells were transfected with pTRE-GFP or pTRE-GFP-TDP_220-414 for 2 days. During these 2 days, cultures were treated with or without doxycycline and MG-132, as indicated. Cells were then harvested for Western blot analysis using anti-GFP and anti-GAPDH. GFP-TDP_220-414, but not GFP, was degraded by the proteasome. Figures shown are representative of the results obtained from 3 independent experiments.