Fig. 3. E4-ORF4 interacts preferentially with hyperphosphorylated SR proteins. (A) SR proteins prepared from HeLa cells (SR-HeLa, lane 1) and adenovirus-infected HeLa cells (SR-Ad, lane 4) were pre-incubated in nuclear extract (HeLa-NE) or nuclear extract depleted of ATP [HeLa-NE(–ATP)] (lanes 2, 3, 5 and 6). Samples were separated on a 10% SDS–polyacrylamide gel, transferred to a membrane and probed with mAb104 (an SR protein-specific antibody that detects phospho-epitopes in the RS domain) in a western blot assay. (B) The filter was probed with mAb96 (an SF2/ASF-specific antibody that detects the RRM1 of SF2/ASF) in a western blot assay. (C) The filter was probed with GST–E4-ORF4 in a far western blot assay. E4-ORF4-interacting proteins were visualized by chemiluminescence using an anti-E4-ORF4 antibody. Note that the autoradiograms shown are taken from different experiments.