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. 2010 May 26;26(14):1714–1722. doi: 10.1093/bioinformatics/btq267

Fig. 4.

Fig. 4.

Immunoblots of predicted GrB substrates. K562 lysates were treated with increasing concentrations of GrB or a mixture of caspase-3 and caspase-8 for either 1 h or 19 h. The final concentration of exogenously-added protease was 1 μ M, 500, 250, 100, 50 and 25 nM. For caspases, the final concentration refers to the concentration of total caspase (caspase-3 plus caspase-8). The no protease controls were incubated at 37○C for 19 h to account for the activity of endogenous proteases. The caspase-inhibited lysates were pretreated with 100 μM z-VAD-FMK and 100 μM z-DEVD-FMK at 37○C and then treated with GrB. Bands corresponding to full-length (FL) protein, proteolytic fragment 1 (PF1) and proteolytic fragment 2 (PF2) are indicated with arrows. Controls showed that the SMN1 antibody cross-reacts with GrB (Supplementary Fig. 4). The GrB band is indicated by an arrow and asterisk.