Fig 2: Splicing is dependent on Tat-associated cellular factors. (A) Down-regulation by siRNA of cellular transcription-splicing factors. HEK-293T were transfected with the reporter construct pLTR-S1Xm-R, pTat and the indicated a control siRNA (si_Cont) or siRNAs to Tat-SF1 (si_Tat-SF1), CA150 (si_CA150), SKIP (si_SKIP), Tat-CT1 (si_Tat-CT1). Transcripts were quantified by RT-qPCR. A RNA sample, which was not reverse transcribed, was utilized as negative control (-RT). (B) Tat transcription transactivation. Fold activation of transcription in the presence of Tat and the indicated siRNAs. (C) RT-PCR analysis of the alternative spliced mRNAs generated by pLTR-S1Xm-R cotransfected with either the control pLuc or pTat and the indicated siRNA (top of the panel). (D) Unspliced (US) mRNAs and the sum of all mRNA transcripts (TOT) are quantified and their ratio is indicated. (E) The splicing products representing splicing to the tat and env specific 3'ss #3 and 5 were quantified and their ratio is indicated. (F) SF2 and SC35 co-immunoprecipitate with CA150 in HeLa nuclear extracts. CA150 was immunoprecipitated with anti-CA150 antibodies and anti-tubulin antibodies as control (indicated on top). Short RNA transcripts containing TAR or a control sequence were added to the assay mixture as indicated. Recombinant GST-Tat protein was also added to the incubation mixture as indicated. The immunoprecipitates were analyzed by western blotting with the indicated antibodies (panels from top to bottom). Quantification of (G) SF2 and (H) SC35 recovered in each immunoprecipitation assays. The arbitrary value of 1 was assigned to the amount recovered by precipitation with anti-CA150 antibodies in the absence of exogenous RNA and Tat. Data were quantified by optical density analysis. Data are represented as means ± SEM.