PARP-14 mediates IL-4 regulation of genes that influence caspase activation, apoptosis, and B lymphomagenesis. (A) Role of PARP-14 in repression of Id-3 by IL-4. RNA from B cells of WT and PARP-14 KO mice, cultured for 20 hours with and without IL-4, was analyzed by Northern blotting for Id-3 and a GAPDH control. An autoradiograph from one such analysis is shown, representative of 3 independent experiments. Below each lane are the PhosphorImager quantitations (in arbitrary units of pixel density) of the relative radioactive signal for each band, and ethidium bromide–stained rRNA signals designated by ◀ (28S, 18S). Note that GAPDH transcription rates and mRNA levels are known to increase in response to IL-4. (B,C) PARP-14 mediates IL-4 induction of the pro-survival kinase Pim-1. B cells purified from WT, PARP-14^−/−, and Stat6^−/− mice were cultured in medium, alone or supplemented with IL-4. After determination of the optimal induction kinetics for each (not shown), Pim-1 (B) or Pim-2 (C) mRNA levels were assayed in RNA isolated after 4 hours (B) or 12 hours (C), using both conventional (left panel) and real-time RT-PCR (right panel). For conventional semiquantitative RT-PCR, the panels show ethidium bromide–stained results from one experiment representative of 4 independent replicates, with the linear relationship of amplifications to template amount documented by using 2 amounts of input cDNA in separate amplifications for each sample (5 × 10⁴ and 1:3 dilution of 5 × 10⁴ cell equivalents of recovered RNA; increasing amounts symbolized by the slope of the black triangle above each lane pair). Southern blotting probed with an internal oligonucleotide sequence was used to confirm the identity, linearity, and relative abundance of the signals (data not shown). To the right in panels B and C, bar graphs show representative data (mean ± SEM; *P < .05) from separate real-time quantitative RT-PCR performed on the independent replicate samples, with each Pim kinase cDNA signal normalized to the quantitative RT-PCR result for hypoxanthine phosphoribosyl transferase cDNA levels in each sample. (D,E) PARP-14 mediates IL-4 regulation of antiapoptotic Bcl-2-family protein. Extracts of purified B cells cultured (20 hours) in the presence or absence of IL-4 were analyzed by immunoblots using anti–Bcl-xL (D) and anti-Mcl-1 (E) Ab, along with antiactin as a loading control. Quantitation of the relative levels of Bcl-xL and Mcl-1 after normalization to the actin controls (each in arbitrary fluorescence intensity units detected by the Odyssey imager) is shown in each bar graph (one representative result from reproducible data [n = 3 independent replicate experiments]). Mcl-1 appears as a doublet because the antibody detects both long (40 kDa) and short (32 kDa) forms expressed in cells.⁴⁸