ADP ribosylation mediates survival signaling in B cells, and PARP-14 has intrinsic PARP activity. (A) Stat6 regulation is intact in PARP-14 KO cells. Splenocytes from WT (+/+) and PARP-14–null (−/−) mice were cultured in media, alone (−IL-4), stimulated with IL-4 (0.5 and 12 hours; +IL-4), or stimulated by IL-4 (30 minutes) followed by removal of IL-4 and further culture for 6 hours. (B) ADP-ribosyltransferase activity associated with PARP-14. Proteins from cells transfected with pcDNA3 or pcDNA3-FLAG-PARP-14 were immunoprecipitated, and bead-bound immune complexes were assayed for transfer of ³²P-labeled ADP-ribose from NAD⁺ onto proteins in the presence or absence of the cell-permeable PARP inhibitor PJ-34 (1 μM). Shown are autoradiographs of the labeled proteins (top panel) and immunoblots to test protein expression levels (bottom panel) after resolution by SDS-PAGE. An arrowhead marks the position on the gel autoradiograph expected for full-length PARP-14 based on the Western blot; the radioactive material marked by a closed circle derived from nonspecific polymeric material generated by active PARP and trapped at the stacker/resolving gel interface. (C) Impaired survival signaling in B cells subjected to PARP inhibition. B cells were pretreated with PJ-34 (1 μM), during culture (20 hours) in the presence or absence of IL-4 and analyzed by TUNEL assays. Shown are FACS profiles in the B220⁺ gate (left, FACS profile) as well as a bar graph showing the mean (± SEM) percentage of cells rescued from apoptosis by IL-4 in the 3 independent replicate experiments (n = 3; *P < .05).