Intracellular association of APO3G and host RNAs. (A) Expression and immunoprecipitation of APO3G. HeLa cells (5 × 106) were transfected with 5 μg of pcDNA-Apo3G-MycHis plasmid DNA. Cells were harvested 24 h post transfection. An aliquot of the transfected cells was used for the analysis of APO3G expression as follows: Cell lysates were immunoprecipitated with a polyclonal antibody to the myc epitope tag (α-myc) or were mock immunoprecipitated (mock). Immunoprecipitated samples and total cell lysate (Total) were analyzed for the presence of APO3G by immunoblotting using an APO3G-specific polyclonal peptide antibody. (B) The remaining cells from above were used for RT-PCR analysis as follows: Total cellular RNA (Total) or RNA present in the immune complexes (α-myc and mock, respectively) was extracted and used for RT-PCR analysis as described in Methods. Primer pairs were selected for the specific amplification of the RNAs as indicated on the left. Primer sequences are listed in table 1. All RT-PCR reactions were performed simultaneously to minimize experimental error. RT-PCR products were analyzed on 1% agarose gels and visualized by staining with ethidium bromide. (C) HeLa cells (5 × 106) were transfected with 5 μg of pcDNA-Apo3G-MycHis plasmid DNA (lanes 1 & 3) or 5 μg of pcDNA-Apo3G (lanes 2 & 4). Cells were harvested 24 h post transfection and analyzed as in panels A and B. (D) The specificity of the RT-PCR reaction was validated using 7SL RNA as a substrate. Total cellular RNA from panel B was either left untreated (-) or treated with RNase A (50 μg/ml) for 60 min at 37°C (+) prior to RT-PCR.