Presence of two distinct signaling pathways regulating Bach2 transcripts and protein. (A and B) Immunoblot analysis (A) or quantitative RT-PCR analysis (B) in Irf4−/− Irf8−/− pre-B cells cultured in the presence of LY294002 under IL-7 Hi conditions for 48 h. (C and D) Immunoblot analysis (C) or quantitative RT-PCR analysis (D) in Irf4−/− Irf8−/− pre-B cells cultured in the presence of rapamycin or AZD under IL-7 Hi conditions for 24 h. For panels A and C, HPRT served as an internal control. IB, immunoblot; p-Bach2, phosphorylated Bach2. For panels B and D, data under IL-7 Lo conditions for 24 h are shown as a control. Relative expression was normalized by Β2M mRNA, and the expression and SEM data were from three independent experiments. *, P < 0.05; ***, P < 0.001. (E) Subcellular localization of Bach2 in Irf4−/− Irf8−/− pre-B cells cultured in the presence of rapamycin. Lamin B1 served as a control for the nuclear fraction, and α-tubulin and HPRT were used as controls for the cytoplasm fraction. WCE, whole-cell extract.