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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: Mol Cancer Ther. 2015 Oct 29;15(1):114–124. doi: 10.1158/1535-7163.MCT-15-0400

Figure 4. Death receptor dual agonist Surrobody is a potent inducer of caspase activation.

Figure 4

MDA-MB-231 cells were cultured in 384 wells (2000 cells per well) and treated with increasing concentrations of DR agonists for 6 hr. The activity of CASPASE 8 (A) and CASPASE 3/7 (B) was assessed using CASPASE-8 Glo (contains IETD peptide) and ApoLive-Glo (contains DEVD peptide) reagents, respectively, from Promega, expressing data as relative luminescence units (RLU) generated per hour per 2000 cells (mean ± SEM; n= 4) (C) MDA-MB-231 cells were plated at a density of 70,000 cells/well and the next day they were treated for 3 h with DR agonists at 1 nM. Before addition to cells, all the monospecific antibodies and Surrobody were incubated for 5 minutes with 0.5× molar ratio of protein G to facilitate clustering. Cells were lysed into SDS-sample buffer and lysates were analyzed by SDS-PAGE/immunoblotting using antibodies specific for cleaved CASPASE-8, cleaved CASPASE-3, PARP, or beta-actin. Molecular weight markers are indicated in kilo-Daltons (kDa). Processed CASPASE-8 large (44/42 kDa) and small (18 kDa) subunits are shown. Processed CASPASE-3 large subunits (17/19 kDa) are shown.