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. 2015 Dec 7;7(3):3461–3476. doi: 10.18632/oncotarget.6505

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Copyright: © 2016 Sarkar et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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A. Immunoprecipitation analysis (3000 μg) with anti-FLAG antibody showing endogenous XIAP levels in MEF cells engineered with WT and Casp3 mutants. 50 μg total extract was loaded as input control (left panel). Arrow indicates IgG heavy chain. B. Co-immunoprecipitation analysis using HEK293 cells transiently transfected with HA-XIAP and indicated Casp3 plasmids. 50 μg total extract was loaded as input control (left panel). Immunoprecipitated proteins were either probed with anti-FLAG antibody (upper panel; showing amount of Casp3 bound to XIAP) or anti-HA antibody showing amount of XIAP bound to Casp3 (lower panel). Arrow indicates IgG light (∼ 25kD upper panel) or heavy (∼53kD lower panel) chains. C. Immunoblot analysis of total protein isolated from stably transfected MEF cells with indicated plasmids showing smac mimetic (066) induced cleavage of Casp3 (250 μg; arrow indicating 17kD cleaved Casp3 band) and loss of full length PARP (250 μg), XIAP and smac (50 μg) protein expressions. GAPDH (50 μg from similar extracts) was probed for loading control. D. Annexin V-PI FACS analysis induced by smac mimetic (066) (n = 5; Mean ± SE) showing relative live cell in the indicated cell clones compared with individual DMSO controls. *Significant difference from control. E. Casp3 and XIAP mRNA expressions and their relative ratios in stably transfected MEF cells expressing vector, WT or Zn-Casp3 mutants. Cells were treated and processed for real-time RT-PCR assays. Data are mean ± SE (n = 3). F. Time course of cycloheximide (CHX) treatment in MEF cells showing XIAP protein expression (upper panel) in control, WT and Zn-Casp3 mutants. GAPDH was probed for loading control. G. Densitometry analysis (n = 5; Mean ± SE) showing relative XIAP protein levels in vector control, single (E272A), double (H108A+E272A and C148S+E271A) and triple (H108A+C148S+E272A) mutants compared with WT Casp3 transfected cells at identical time points. *Significant difference from control.