FIG. 3.

CTIP2 and HIV-1 Tat interact in vitro and in cells. (A) Schematic representation of the domains present in the viral Tat protein. (B) The N-terminal region of Tat interacts with CTIP2. GST pull down assays were performed with ³⁵S-CTIP2 and the indicated GST-Tat fusion proteins. ³⁵S-CTIP2 was translated in vitro and incubated with GST (lane 2) or the indicated full-length and truncated GST-Tat proteins (lanes 3 to 5). After extensive washing, the bound proteins were eluted and analyzed by SDS-polyacrylamide gel electrophoresis. Lane 1, input ³⁵S-CTIP2 (0.2 μl); lanes 2 to 5, GST and GST-Tat incubated with ³⁵S-CTIP2 (10 μl). (C) SDS-PAGE of GST and GST-Tat fusion proteins used in panel B visualized by Coomassie brilliant blue staining. (D) Tat interacts with CTIP2 in vivo. Nuclear protein extracts from microglial cells, previously transfected with CTIP2 in the absence or presence of pCMV-Tat were immunoprecipitated with monoclonal anti-Tat antibodies (lanes 3 and 4). The presence of CTIP2 in the nuclear extracts (lanes 1 and 2) and in the immunoprecipitates (lanes 3 and 4) was detected with anti-Flag antibodies.