Figure 2: Calpain-dependent cleavage of TDP-43 in vitro.
From: A role for calpain-dependent cleavage of TDP-43 in amyotrophic lateral sclerosis pathology
(a) Neuro2a cell lysates were analysed by western blotting with TDP-43 antibodies after treatment with calpain-I (Calp-I), caspase-3 (Casp) and/or CaCl2 (Ca2+). Calpain cleaved TDP-43 into fragments with an apparent molecular mass of 33–36 kDa, whereas caspase-3 failed to cleave TDP-43 at the concentration that effectively cleaved α-spectrin. The activation of calpain and caspase-3 was demonstrated by the cleavage of α-spectrin into calpain-dependent spectrin breakdown products (SBDP) (150b and 145) and caspase-3-dependent SBDP (150a and 120). (b,c) Levels of recombinant human TDP-43 declined in the presence of calpain-I in a time-dependent (b) and a concentration-dependent manner (c). (d) Calpain-II cleaved recombinant human TDP-43 in the same manner as calpain-I. (e) Inhibitors (inh) of calpain (MDL28170, ALLN and leupeptin) but not inh of other proteases (phenylmethylsulphonyl fluoride (PMSF), aprotinin, pepstatin, roscovitine and Z-VAD-fmk) effectively inhibited the calpain-dependent cleavage of TDP-43 in Neuro2a cell lysates. Arrows, full-length proteins.
