Role of CUGBP1 in imatinib-induced CEBPB expression. (A) Levels of expression of CUGBP1 and CEBPB proteins assessed by Western blotting in parental (lane 1) or 32D-BCR/ABL-expressing cells untreated or treated with 1 μM STI571 (lanes 2-4). Specific antibodies against C/EBPβ or CUGBP1 were used to probe the nitrocellulose filter. (B) Histogram (top) and Western blot (bottom) show CUGBP1 levels (expressed in the graph as mean ± SE of densitometric units after normalization with GRB2 levels) in the CD34⁺ fraction from NBM (n = 3), CML-CP (n = 3), and CML-BC (n = 3) marrow samples (NBM versus CML-CP, *P = .272; CML-CP versus CML-BC, **P = .003; t test). GRB2 levels were measured as loading control. (C) Effects of CUGBP1 on C/EBPβ expression in GFP-sorted 32D-BCR/ABL cells or puromycin-selected K562 cells. Western blot shows CEBPB levels in empty vector-transduced cells (lanes 1 and 3) or in cells ectopically expressing CUGBP1 (lanes 2 and 4). Exogenous CUGBP1 was detected by anti-Flag Western blotting. (D) Immunoprecipitation with anti-Flag–conjugated agarose beads: cytoplasmic extracts (lanes 1 and 4) from GFP-sorted cells carrying the empty vector (lane 4), or expressing CUGBP1-Flag (lane 1) were UV–cross-linked and immunoprecipitated after incubation with ³²P-labeled oligo-RNAs βLAPu (lanes 3 and 5) or sORFβ (lane 2). Cytoplasmic extracts (lanes 1 and 4) and immunoprecipitates (lanes 2, 3, and 5) were resolved by SDS-PAGE and then probed in Western blotting by an anti-Flag epitope antibody (top panel); the filter was then exposed to an x-ray film to detect the radioactive signal of the RNA-protein complex (bottom panel).