Expression and subcellular distribution. A, FLAG-tagged TRIM5α (rhesus, human, or African green monkey orthologues) and TRIMCyp were stably expressed in MDTF and in D17 cells, and expression was assessed by western blotting with antibodies directed against FLAG (top) or actin (bottom). The percentage of transduced cells was roughly similar for all cell lines created, as judged by the percentage of puromycin-resistant cells (not shown). The presence of the FLAG tag in TRIM5αhu was confirmed by sequencing of the plasmid DNA. B, MDTF or D17 cells expressing TRIM5αrh or TRIMCyp were fixed and stained using an antibody against FLAG and counterstained with Hoechst33342 to reveal DNA.