Fig. 5: Caspase-2 directly interacts with p54nrb and cleaves p54nrb at D422.

A Immunoblot of p54nrb caspase-2 and caspase-3 after endogenous immunoprecipitation (IP) of p54nrb from HeLa cells. B Immunoblot of p54nrb and caspase-2 after in vitro caspase cleavage assay with 0.2 µg or 2 µg recombinant p54nrb and 10 U human recombinant active caspase-2 after 3 h incubation at 37 °C. C Immunoblot of p54nrb cleavage and caspase-2 from HeLa cells at 48 h after ectopic expression of 1 µg/ml empty vector pcDNA3-Flag, pcDNA3-caspase-2-Flag, and pcDNA3-caspase-2-C303A-Flag (inactive). D Graphical illustration of p54nrb cleavage by activated caspase-2 via either overexpression, apoptotic induction, or recombinant caspase-2 protein. The active site of caspase-2 dimer is marked with yellow stars. Location of the putative cleavage sites in the p54nrb protein domains are marked with arrows, and its anticipated cleavage fragments are shown. E Immunoblot of p54nrb cleavage in HeLa shRNA-p54nrb#1 cells after ectopic expression of pcDNA3-Flag, pFlag-p54nrb, pFlag-p54nrb-D58N and pFlag-p54nrb-D422A (1 µg/ml plasmid) (+24 h) and 24 h treatment with 50 µM Eto. F Endogenous ribonucleoprotein immunoprecipitation of p54nrb in HeLa cells and subsequent isolation of coprecipitated DNA or RNA coupled with cDNA-synthesis. Validation of potential co-precipitate by PCR and subsequent agarose gel-electrophoretic detection. G In vitro p54nrb/DNA binding assay. 0.5 µg human recombinant p54nrb (Origene, Rockville, MD USA) and 100 ng plasmid encoding the sequence of gelsolin (Ch-gelsoln) [#37262] [Addgene]) were incubated at 37 °C for 18 h. Immunoprecipitation (IP) was performed by employing either p54nrb antibody or the same amount and species of IgG as control. Next, the potentially binding DNA was isolated as described in Methods (see ChIP). P54nrb was detected by western blot and the binding of specific DNA was confirmed by PCR, employing gelsolin-specific primers.