Fig 1: The SR proteins SRSF2 and SRSF6 bind downstream of 3'ss A3 to control tat -mRNA splicing. (A) In vitro-transcribed RNA substrates used for the RNA pull-down assays. Mutated nucleotides are indicated below the wild-type reference (pNL4-3) at corresponding positions and “-“ denotes wild type nucleotide. (B) RNAs were immobilized on Agarose beads and analyzed for the presence of SR proteins with specific antibodies directed against SRSF2 (Abcam, ab28428) or phosphorylated SR proteins (Invitrogen, 1H4G7). Recombinant MS2 coat protein was added to HeLa cell nuclear extracts and served as a control for equal precipitation efficiencies. (C) 2.5 × 105 HEK293T were transfected with pNL4-3 or mutant provirus and pcDNA3.1(+), an SRSF2 or SRSF6-expressing plasmid. 48 h after transfection RNAs were analyzed by RT-PCR (C) or Northern blot (D). (E) Cell lysates and supernatants from transfected HEK293T cells were analyzed for Gag expression as described before (see Figure 2). (F) Left: Cell lysates and supernatants from HEK293T cells cotransfected with pNL4-3 and gradually increasing amounts of SRSF6 expressing plasmid that were analyzed for Gag expression as described before. Right: Serial dilutions of cell lysates and supernatants from transfected HEK293T cells analyzed for Gag expression.