Fig 5.
Binding of TRIM5αrh RING domain variants to the HIV-1 CA. 293T cells were transfected with plasmids expressing the indicated wild-type and mutant TRIM5αrh proteins tagged with HA epitopes. Thirty-six hours after transfection, cells were lysed. The lysates were incubated at room temperature for 1 h with HIV-1 CA-NC complexes that had been assembled in vitro. The mixtures were applied onto a 70% sucrose cushion and centrifuged. INPUT represents the lysates analyzed by Western blotting before being applied to the 70% cushion. The input mixtures were Western blotted using anti-HA antibodies. The pellet from the 70% cushion (BOUND) was analyzed by Western blotting using antibodies against the HA tag and HIV-1 CA-NC protein. The blots were quantitated as described in Materials and Methods, and values for binding are shown in Tables 1 and 2. The results of three independent experiments were similar; the result of a single experiment is shown.