Skip to main content
. Author manuscript; available in PMC: 2013 Sep 1.
Published in final edited form as: Biochimie. 2012 May 29;94(9):1974–1981. doi: 10.1016/j.biochi.2012.05.020

Figure 3. Stimulation of caspase-7 mediated cleavage of TDP-43 by 4-aminoquinolines is due to a specific interaction with TDP-43.

Figure 3

(A) Time dependence of 2 stimulation of caspase-7 mediated cleavage of TDP-43. TDP-43 (2 μg/mL) was preincubated with 10 μM of 2 for 1 hr followed by the addition of caspase-7 (5 U/mL). Reactions were incubated at 37 °C and terminated at the times indicated with SDS sample buffer followed by immunoblotting (section 2.3). (B) TG12 mediated antagonism of 2 stimulation of caspase-7 mediated cleavage of TDP-43. TDP-43 (2 μg/mL) was preincubated for 1 hr with indicated concentrations of 2 in the presence and absence of 1 μM TG12 followed by the addition of caspase-7. Reactions were terminated after 1 hr at 37 °C with SDS sample buffer. (C) Stimulation of caspase-7 cleavage of TDP-43 requires correctly folded TDP-43. TDP-43 (0.2 mg/mL) was incubated in the absence and presence of 6 M guanidine HCl for 1 hr, followed by dialysis for 3 hr at room temperature. Dialyzed TDP-43 was then diluted to 4 μg/mL and preincubated in the presence and absence of 2 for 1 hr followed by addition of caspase-7 and subsequent 1 hr incubation at 37 °C. Blots are examples of single experiments that were replicated with similar results. (□) TDP-43, (◇) TDP-43 CTF35, (○) nonspecific band. (D). 4-aminoquinoline 2 does not affect caspase-7 mediated cleavage of Ac-DEVD-AMC. Caspase-7 (4 U/mL) was incubated with 1 μM Ac-DEVD-AMC in the presence and absence of indicated concentrations of 2 and change in fluorescence was monitored as described in section 2.4. Data represent the mean ± S.E.M of four determinations.