Figure 8
Enzymatic activity ofEhPRMT1a and co-localization of EhPRMT1 and EhR8me2. (A) Recombinant EhPRMT1a (1 μg) was incubated with10 μg of the nuclear basic fraction of E. histolytica trophozoites (NF) or with 0 to 4 μg of chicken histones in the presence of [3H]AdoMet and incorporation of radioactivity on the substrates was analyzed by scintillation counter. As a negative control, 1 μg of lysozyme (L) was incubated with 10 μg of the nuclear basic fraction or with 4 μg of chicken histones. Results are showed as the mean value ± standard deviation of three assays performed by duplicate. (B) 1 μg of recombinant EhPRMT1a or lysozyme (negative control) were incubated with 4 μg of chicken histones in the presence of non-radioactive AdoMet. Then, substrate specificity of EhPRMT1a was analyzed by Western blot using α-H4R3me2. Left: 15% SDS-PAGE of chicken histones stained with Coomassie blue. Right: Western blot showing the detection of H4R3me2. Under is shown the alignment of the N-termini of chicken (Gg) and E. histolytica (Eh) H4 histones. (*) Identical residues. (.) Similar residues. Arrow indicates the arginine residue modified by PRMT1. (C) α-H4R3me2 and α-PRMT1 antibodies were labeled with Pacific blue 410 (green) and Alexa fluor 647 (red) respectively. Then, E. histolytica trophozoites were processed for immunofluorescence assays using these antibodies. Nuclei were stained with DAPI (blue). Right panels show a magnification of a single trophozoite. Zy planes, lateral images of the cells.