Figure 2
Time-dependent expression and aggregation of GFP-TDP 220-414 in M17D3 cells. M17D3 cells were cultured in media lacking or containing 1 μg/ml doxycycline to either induce or suppress the expression of GFP-TDP220-414, respectively. (A) The expression of total and phosphorylated-GFP-TDP220-414 were assessed using antibodies towards the C-terminus of TDP-43 (cTDP-43) or anti-pTDP-43, respectively. Endogenous TDP-43 is indicated by an asterisk (*) while dashed arrows indicate TDP-43 fragments, which likely resulted from the truncation of GFP-TDP220-414. HMW=high-molecular weight, NS = non-specific band, Dox=doxycycline. (B) GFP fluorescence in cells expressing GFP-TDP220-414 was viewed by fluorescent confocal microscopy at 2, 4 and 6 days post-induction and in non-induced controls. Scale bar, 20 μM. (C) GFP-TDP220-414-expressing cells were immunostained with antibodies towards S409/S410-phosphorylated TDP-43 or ubiquitin. Scale bar, 10 μM. Figures shown are representative of the results obtained from 4 independent experiments. (D) M17 founder cells were cotransfected for 48 h with HA-ubiquitin and GFP or GFP-TDP220-414. Cell lysates were incubated with anti-GFP. Protein G agarose was added to capture the protein-antibody complex and then the captured protein was eluted from the beads and resolved by SDS-PAGE for Western blot analysis. Blots were probed with antibodies towards the HA-tag or GFP. Figures shown are representative of the results obtained from 3 independent experiments.