Fig 2: Overexpression of SRp20, Tra2ß1, or Tra2ß3 suppress HIV-1 expression.(A) Diagram of SRp20, Tra2ß1, and Tra2ß3 protein domain structure. (B) Cells were transfected with the vectors CMVmyc (mock), CMVmyc SRp20 (SRp20), CMVmyc Tra2ß1 (Tra2ß1), or CMVmyc Tra2ß3 (Tra2ß3). Forty-eight hours post-transfection, cells were harvested, lysed in RIPA buffer, and proteins analyzed by western blot with anti-myc antibody followed by anti-tubulin (Tubulin) antibody to indicate loading. (C) HeLa rtTA-HIV-?Mls cells were transfected with (+) (or without, -) CMVtTApA (tTA) to activate the endogenous HIV-1 provirus along with CMV PLAP and either CMVmyc, CMVmyc SRp20, CMVmyc Tra2ß1, or CMVmyc Tra2ß3. Forty-eight hours post transfection, cell media was harvested and assayed by p24CA ELISA for expression of HIV-1 Gag (black) and production of SEAP (white). Results are averaged from 6 independent assays, error bars are SEM, and asterisks indicate values deemed significant from CMVmyc (+tTA) as detailed in “Materials & Methods”. (D) In parallel, total RNA was extracted from transfected cells and the abundance of individual viral RNA (unspliced, US; singly spliced, SS; multiply spliced, MS) were determined by qRT-PCR. Shown are results averaged from 6 independent assays, error bars are SEM, and asterisks indicate significance as noted above. (E, F) MS HIV-1 RNAs from transfected cells were amplified by RT-PCR as described in Fig. 4. Amplicons were fractionated on urea-PAGE gels and a representative gel is shown (E). Relative abundance of individual MS viral RNA species were quantitated and graphed (F). Data was averaged from >5 independent assays, errors bars are SEM, and asterisks indicate statistical significance as described above.