Fig. 4.

DDX17 interacts with essential splicing factors. (a) Schematic representation of HIV-1 genome organization and mapping of the splice sites described in this manuscript. Other known sites have been omitted for simplicity. (b) Exogenously expressed Myc-DDX17 co-immunoprecipitates with endogenous U2AF65, SRSF1/SF2 and U1-C. IgG lane, immunoprecipitation of proteins treated with isotype control matched antibody. In RNase-treated lane, samples were subjected to digestion with RNase A. (c) HeLa M cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI, with or without increasing concentrations of siDDX17 rescue expressor construct. Expression of U2AF65, SRSF1/SF2 and U1-C, following knockdown and rescue of DDX17 was detected by Western blotting, using their respective antibodies. GAPDH was detected to monitor sample loading. (d) Effect of overexpression of DDX5 as attempted rescue of HIV-1 virus production following endogenous DDX17 depletion. Cells were sequentially transfected with siControl or siDDX17 and then a second round of siRNA together with pLAI, with or without increasing concentrations of DDX5 expressor construct. (e) Western blot for DDX17 and DDX5 expression. The graph is a representative of three independent experiments done in triplicate. Bars represent mean of triplicate samples ± SEM. Statistical significance: **P < 0.01.