(A) Immunoblot analysis of GST pull-down reactions involving recombinant Sam68 and GST-fusion proteins of U2AF65 and U2AF35. The GST protein served as a control. Immunodetection with anti-Sam68 and anti-GST antibodies is shown in the upper and lower panels, respectively. Black bars on the left indicate positions of marker bands. (B) Co-immunoprecipitation from LB17-lymphoma cell lysates with an anti-Sam68 antibody or a corresponding control antibody (CoAb). In lane 3, the lysate was loaded. Precipitates were analysed by immunoblotting with an anti-U2AF65 antibody. Arrows indicate U2AF65 band; asterisks denote bands caused by immunoglobulins. (C) Co-immunoprecipitation experiment as in (B), in the presence (+) or absence (−) of RNase A. Symbols are as in (B).

(A) Immunoblotting of non-phosphorylated (−) and ERK-phosphorylated (P) recombinant Sam68 preparations used in (B). Phosphorylation of Sam68 at ERK target sites results in a molecular weight shift (arrow) [19]. Black bar on the left indicates marker band. (B) Electrophoretic mobility shift assay using RNA oligonucleotide probes comprising the exonic or intronic Sam68 binding sites and different amounts of either non-phosphorylated (−) or phosphorylated (P) recombinant Sam68.

(A) Schematic drawing of the examined CD44 minigene pre-mRNA region (CD44 exon v5, open box; upstream intron, line; Sam68 binding sites, black oval and box, respectively; arrows, PCR primer). (B) RNP immunoprecipitations with an anti-U2AF65 antibody or a control antibody (CoAb) from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct containing CD44 exon v5. Cells were left either untreated (−) or treated with phorbol ester (TPA). TPA treatment and RNP immunoprecipitations were performed as described in legend to Figure 4. (C) RNP immunoprecipitations of U2AF65 from LB17 lymphoma cells co-transfected with the pETv5 minigene construct and either a Sam68 expression plasmid (+) or the empty expression vector as a control (−). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.

(A) Scheme indicating positions of the Sam68 binding sites (black oval and box, respectively) in CD44 exon v5 (open box) and the upstream intron (line). The Sam68 consensus binding site in the wild-type RNA sequence used for the electrophoretic mobility shift assay (EMSA) in panel B is boxed. The mutated nucleotide of the A/C mutant is indicated in red. (B) EMSA using recombinant Sam68 and radioactively labeled RNA oligonucleotide probes comprising either the wild-type or the A/C-mutant version of the intronic Sam68 binding site (see panel A). (C) v5-luciferase fusion activity from LB17 lymphoma cells transfected with different pETv5luc splice-reporter genes [15]. They were mutated for either the exonic (L/CC mutant [19]), the intronic (A/C mutant), or both Sam68 binding sites (double mutant). Cells were treated with 12-o-tetradecanoylphorbol-13-acetate (TPA, 40 ngml⁻¹) (+) or with DMSO (solvent control) (−) for 6h prior to lysis and measurement of luciferase activity. Error bars indicate standard deviations from three independent transfections.

(A) Detection of Sam68 binding to an endogenous CD44 pre-mRNA fragment (spanning the v5 exon and 100 bp upstream intron sequence) in LB17 lymphoma cells by RNP immunoprecipitations. A control antibody (CoAb) or an anti-Sam68 antibody were used for precipitations. Lower panels show amplifications from the lysate used as input for the precipitations. Bands correspond to the expected size of 228 bp. Positions of primers used for amplification are indicated as arrowheads in scheme on top; Sam68 binding sites (black oval and box, respectively), CD44 exons v4 and v5 (open boxes), intron (line). –RT represents reactions without reverse transcriptase. (B) RNP immunoprecipitations with either a control antibody (CoAb) or an anti-Sam68 antibody from lysates of LB17 lymphoma cells that were transfected with the pETv5 minigene construct [23]. The cells were either left untreated (−) or treated with 40 ng/ml of 12-o-tetradecanoylphorbol-13-acetate (TPA) for 10 min. A fragment from the minigene pre-mRNA spanning the v5 exon and 450 bp upstream intron sequence (In-v5) was amplified using the primers indicated (scheme on top; symbols as in panel A). Specific amplification of the minigene pre-mRNA is achieved by the forward primer that is complementary to the deletion junction of the v4 exon in the construct. To check for potential differential loss of material during the precipitation and washing procedure, we monitored the amounts of GAPDH mRNA unspecifically bound to the antibody and/or the agarose beads (lower panel). Bands correspond to the expected sizes of 600 bp (In-v5) and 278 bp (GAPDH). All PCR amplifications were in linear phase as verified with different amounts of cDNA.