Fig. 2

The effect of Ku70 on HIV-1 integrase stability in 293T cells. a Immunoprecipitation of HIV-1 IN_HA on anti-HA antibody conjugated beads (middle panel) or of Ku70_3×FLAG on anti-FLAG antibody conjugated beads (lower panel). IN_wt or IN_mut were expressed in 293T cells with Ku70_3×FLAG or with an empty vector, then cells were lysed, 10% of lysates were saved for input analysis (upper panel), the rest was divided in two and subjected to immunoprecipitation for 4 h at 4 °C, then the beads were washed 4 times with incubation buffer and proteins were eluted by 0.1 M glycine, pH 2.5, and the eluates together with input samples were analyzed by Western blot. Representative images of at least three independent experiments are shown. b Western blot analysis of a superexpression of IN_wt (lanes 2–4) or IN_mut (lanes 5–7) either in the presence of superexpressed Ku70_3×FLAG (lanes 3 and 6) or siRNA for Ku70 (lanes 4 and 7) or an empty vector (lanes 2 and 5). Representative image of at least three independent experiments is shown. c Plot showing IN stability in 293T cells upon cycloheximide treatment. IN relative amount was determined as band intensity normalized to tubulin. Mean values ± SD (n = 3) are shown. The weight marker mobility levels are presented to the right of the WB panels