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. 2015 May 21;6(5):e1769. doi: 10.1038/cddis.2015.132

Figure 3.

Figure 3

Ionomycin induces intracellular Met cleavage by calpains. MCF-10A cells were grown for 24 h, serum starved and treated with (a) 20 μM cathepsin inhibitor Z-FA-FMK, calpain inhibitor calpeptin, or pan-caspase inhibitor QVD or (b) increasing concentrations of calpeptin. They were then treated for 1 h with 1 μM ionomycin (iono). (c) MCF-10A cells were grown for 24 h, serum starved, and treated for 1 h with 1 μM ionomycin or for 6 h with 1 μM staurosporine (stauro). (d) MCF-10A cells were transfected with the same amount of siRNA. The siRNAs tested were as follows: control siRNAs, siRNAs targeting Met or calpain 1 or calpain 2, or a mix of siRNAs targeting calpain 1 and calpain 2. On the next day, they were serum starved overnight and treated for 1 h with 1 μM ionomycin. (a–d) Cell lysates were analyzed by western blotting with an antibody directed against either the kinase domain of human Met, calpain 1, calpain 2, cleaved caspase 3, or GAPDH to assess loading. Arrows indicate the respective positions of the detected proteins and their cleaved forms. Sequential reprobing with calpain 1 and calpain 2 resulted in detection of both proteases (d)