Human and rhesus macaque TRIM5α are SUMO substrates. A. In vitro translated human or rhesus macaque TRIM5α proteins were incubated with purified recombinant E1 (SAE1 and SAE2 subunits, 370 nM), E2 (Ubc9, 630 nM) enzymes, recombinant SUMO1 or SUMO2 (7 μM), and increasing doses of GST-PIAS1 (0, 0.3 or 1.5 μM) or GST alone (0.3 or 1.5 μM) as control. Reaction products were analyzed by 10% SDS-PAGE. B. HeLa cells expressing HA-tagged hTRIM5α or rhTRIM5α were transfected with Ubc9 and 6xHis-SUMO1, when indicated (+). Cell lysates were directly analyzed by SDS-PAGE and immunoblotted with anti-HA antibodies. Alternatively, cell extracts were subjected to an immobilized-metal affinity chromatography (IMAC) on nickel-agarose beads under strongly denaturing conditions and 6xHis-SUMO conjugated proteins were analyzed by SDS-PAGE and immunoblotted with anti-HA antibodies. The position of unmodified TRIM5α is indicated by an asterisk whereas black arrows indicate the position of SUMOylated forms.