Figure 5.

Beclin-1 proteolytic cleavage is abrogated by calpain knockdown. (a) Effectiveness of calpain small-subunit (CAPNS1) silencing. In vivo knockdown of calpains by intravitreal injection of a specific CAPNS-siRNA was verified by the abrogation of the calpain proteolytic activity on α-spectrin in animals subjected to 50 min of ischemia, followed by 1 h of reperfusion. Western blotting analysis showed no changes of calpain proteolytic activity in the retinas from animals treated with the scramble non-targeting (NT) siRNA. Densitometric analysis of the 150/145-kDa SBDP bands showed a reduction by 87% in the CAPNS1 siRNA-treated animals (CAPNS1-siRNA=0.13±0.11 versus vehicle=1±0.02). (b) Beclin-1 cleavage was completely abrogated in the CAPNS1-silenced retinas subjected to ischemia, as shown by the absence of the 50-kDa proteolytic fragment and the recovery of the full-length protein observed at 1 h of reperfusion. Histograms show the densitometric analysis of the autoradiographic bands. Each value is the mean±S.E.M. of three independent experiments. ^***P<0.001 versus vehicle and NT siRNA. L, left eye; MW, molecular weight; R, right ischemic eye