Activation of the m-calpain isoform in the ACT-treated cells.

<p>Western blot of the cytosolic fraction isolated from J774A.1 cells treated with ACT (35 nM) or from cells transfected with anti m-calpain siRNA and treated with ACT (35 nM) (A). Kinetics of the proteolytic cleavage of the FRET-based calpain specific substrate for ACT-treated cells (35 nM toxin), and cells in which m-calpain expression has been silenced by transfection with the corresponding siRNA, or mock cells transfected with scrambled control siRNA, or cells pre-incubated with SAJ6017 (78 nM), a specific m-calpain inhibitor. The figure is a representative experiment from three similar assays performed under identical conditions (B). Assay of calpain activity using FITC-labelled α-casein as substrate. FITC-α-casein hydrolysis by calpain is determined from in J774A.1 cell extracts incubated with FITC-labelled α-casein in the presence or absence of 1 mM CaCl₂. Protease activity was quantified in 10% polyacrylamide gel by determining fluorescence intensity of the non-hydrolysed FITC-α-casein band. Non-digested FITC-α-casein and cell extracts without calcium were used as negative controls (C).