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.

Polyclonal antiserum reactive with EndoU was generated using purified recombinant EndoU protein (347 amino acids, 107-454) fused at the C terminus with a 6-His tag and produced in the BL21 strain of E. coli. EndoU protein was affinity-purified on His-agarose, yielding a single band of correct size on SDS-PAGE gels. Rabbits were immunized and boosted with EndoU protein in adjuvant (Maine Biotechnology Services, Inc.). Anti-EndoU Ab was affinity-purified from immune sera using recombinant EndoU protein covalently bound to agarose beads. Purified spleen B cells or CD4 + T cells (CD4 + T Cell isolation kit; Miltenyi Biotec) were cultured with F(ab) 2 anti-IgM Abs for B cells or CD3e mAb for T cells (16.7 µg/ml, clone 500A2; BD) for 18 h. 10 6 cell equivalents of detergent lysates were separated by SDS-PAGE, followed by transfer to nitrocellulose membranes, with EndoU detected using anti-EndoU Ab, HRP-conjugated donkey anti-rabbit IgG (H+L) secondary Ab (Jackson ImmunoResearch Laboratories), and a Pico Enhanced Chemiluminescence kit (Pierce). The blots were stripped and reprobed with a polyclonal ERK2 Ab (C-14; Santa Cruz Biotechnology, Inc.) to ensure equivalent sample loading. Thymocytes (1.5 × 10 6 cell equivalents/lane) were also analyzed, with the blots reprobed using either polyclonal ERK2 Ab or polyclonal Lck Ab (2102, Santa Cruz Biotechnology, Inc.).