Figure 7
IN complex II depends on LEDGF/p75. (A) LEDGF/p75-knock-down SupT1 cells (TL34) and control SupT1 cells (TC3) were infected with VSV-G a pseudotyped HIV-1Δenv-Luc. 48 h p.i. cell lysates were analyzed for luciferase activity. Control polyclonal SupT1 cells (TC3) (B) or LEDGF/p75 knock-down TL34 cells (C) were infected with HIV-1IN-HA. WCE were prepared at 2 h p.i. and subjected to gel filtration on a Superdex 200HR 10/300 column. Fractions were collected and analyzed by Western blotting using antibodies against LEDGF/p75, TNPO3 and HA. Inputs represent 3,5& of WCE load. IN complexes I and II were detected in both TC3 and TL34 cells; yet a third complex with a lower molecular weight is detected only in TL34 cells (IN complex III, ~150KDa). Dividing lines indicate the grouping of parts of the different gels with identical times of film exposure. (D) Fractions 30-32, corresponding to part of the IN complex III, were pooled and proteins were cross-linked with increasing concentrations (0.25 to 2 mM) of the cross-linker ethylene glycol bis-succinimidylsuccinate (EGS). Cross-linked products were separated by SDS-PAGE and immunoblotted with anti-HA-HRP antibody.