EndoU is a novel regulator of AICD during peripheral B cell selection

Poe, Jonathan C.; Kountikov, Evgueni I.; Lykken, Jacquelyn M.; Natarajan, Abirami; Marchuk, Douglas A.; Tedder, Thomas F.

Flow cytometry analysis. Assessment of cell surface Ags was performed by flow cytometry using fluorochrome-conjugated Abs as previously described (Poe et al., 2004) . For intracellular c-Myc and PCNA expression, purified spleen B cells were cultured for 18 h in the presence or absence of F(ab) 2 anti-mouse IgM Ab and then fixed and permeabilized using a Cytofix/ Cytoperm Plus kit (BD). Intracellular protein staining used either c-Myc Ab (IgG1 clone 9E10; BioLegend), followed by RPE-conjugated goat anti-mouse IgG1 secondary Abs (SouthernBiotech), or PCNA Ab (FITC-conjugated mouse IgG2a clone PC10; eBioscience). Immunofluorescence staining was assessed by flow cytometry, with background staining levels determined using nonreactive isotype-matched, fluorochrome-conjugated control Abs. Asterisks indicate conserved residues within similar functional groups. Numbers indicate the residue positions within mouse EndoU. The positions of the somatomedin B and XendoU domains are shown for reference. (B) Phylogenetic gene tree comparison of EndoU and its orthologs in eukaryotes, prokaryotes, and members of the coronaviruses. The neighbor-joining method was used to construct a phylogenetic tree using outgroup sequences for mouse endoribonucleases Ern1 and Dicer1. The outgroups were removed from the diagram for clarity. Bootstrap values from 1,000 replications are noted near branches. (C) Gene sequence homology between mouse and human EndoU. The region of mouse Chr 15 containing the entire EndoU locus was analyzed against the human genome using the VISTA Browser whole genome comparative analysis tool (http://genome.lbl.gov/vista/index.shtml), with a calculation window and minimum conservation width of 100 bp. Homologous regions of >50% identity are indicated by colored peaks, with predictions of exons, the 3-UTR, and conserved noncoding regions as indicated. For reference, the cartoon graphic above the bottom panel shows the actual positions of EndoU exons and the 3-UTR to scale.