Figure 4
Knockdown of heat shock proteins leads to the accumulation of TDP-43 C-terminal fragments. (A) Co-immunoprecipitation studies showed that GFP-TDP220-414, but not GFP, binds to Hsp70 and Hsp90. M17 founder cells expressing HA-ubiquitin and GFP or GFP-TDP220-414 were harvested and lysates were incubated with anti-GFP. Protein G agarose was added to capture the protein-antibody complex and the captured protein was eluted from the beads and resolved by SDS-PAGE for Western blot analysis. Blots were probed with antibodies towards GFP, Hsp70 or Hsp90 (note that blots were also probed with an antibody toward the HA-tag, which is shown in Figure 2D). Figures shown are representative of the results obtained from 3 independent experiments. (B-C) To explore the role of Hsp70 and Hsp90 in the degradation of GFP-TDP220-414, M17D3 cells were grown in doxycycline-free medium for 4 days then treated with siRNA targeted to Hsp90 or Hsp70 or with a validated negative siRNA control. After 48 h, cells were harvested for Western blot analysis using the indicated antibodies. Levels of total TDP-43, phosphorylated TDP-43, Hsp70 and Hsp90 were determined by densitometric analysis (C). Data was collected from 3 separate experiments and shown as the mean ± SEM. ** represents P < 0.001, as assessed by 1-way ANOVA, followed by Tukey's posthoc analysis.