Figure 2

Detection of transiently expressed CAN1 and CAN2 nuclease activity. The protein extract from protoplasts transformed with empty vectors (pSAT6A-ECFP-N1) was used as a negative control (contr.). (A) Western blot analysis of HA-tagged CAN1 and CAN2 nucleases with anti-HA antibody. Molecular mass deduced from gel electrophoresis as indicated by the arrowheads. (B) Detection of CAN1 and CAN2 DNase activities using the in-gel nuclease activity assay. Type of DNA substrate (ssDNA, dsDNA), ions and EDTA added to the reaction buffers, as indicated. (C) RNase activities of CAN1 wild type and CAN2 wild type (wt) and its mutant lacking anti-codon binding-like domain (Acod) are shown. (D) Western blot analysis of wild type (wt) and ECFP-tagged (C-ECFP) CAN1 and CAN2 proteins with anti-GFP antibody. (E) Detection of wild type (wt) and C-terminal ECFP-tagged (C-ECFP) CAN1 and CAN2 DNase activity using in-gel nuclease activity assay. (F) The deletion mutants of CAN2 lacking the conserved regions of domains described in the text. (Myr/pal.) - myristoylation and palmitoylation motif. (ABC t.) - ABC transporter-like sequence. (N-SNc) - N-terminal part of SNc domain. (C-SNc) - C-terminal part of SNc domain. (Acod.b.) - anti-codon binding-like domain. Numbers in the brackets below the domain name abbreviations indicate the ranges of amino acids removed from each mutant. (G) Western blot analysis of CAN2 mutants with an anti-GFP antibody. The abbreviations of the mutated domains as above (Figure 2F). (H) Detection of the nuclease activity of CAN2 mutants. The abbreviations of mutated domains as above (Figure 2F).