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Figure 7 | Retrovirology

Figure 7

From: Phosphorylation of HIV-1 Tat by CDK2 in HIV-1 transcription

Figure 7

A , CDK2 associates with Tat in cultured cells. Flag-tagged Tat was expressed in HeLa cells by Adeno-Tat infection and precipitated with anti-Flag antibodies from cellular lysate. Co-precipitated proteins were resolved on 12% Tris-Tricine PAGE and analyzed by immunoblotting analysis with anti-CDK2 and anti-Tat antibodies. Lane 1, input control without Tat. Lane 2, input control with Tat. Lanes 3 and 4, extract without or with Tat precipitated with anti-Flag antibodies. Lane 5, extract with Tat precipitated with preimmune mouse IgG. The position of light chain of IgG recognized in anti-Flag immunoprecipitates by anti-mouse HRP-conjugated secondary antibodies is indicated by asterisk. B , CDK2-specific siRNA inhibits association of Tat with CDK2. Flag-tagged Tat was expressed in 293T cells by transfection (lanes 2 and 4). Cells were transfected with non-targeting (lanes 1 and 2) or CDK2-specific (lanes 3 and 4) siRNAs. Lysates were precipitated with anti-Flag antibodies, resolved on 12% Tris-Tricine PAGE and immunoblotted with anti-Tat, anti-CDK9 or anti-CDK2 antibodies. C , CDK2-specific siRNA does not affect association of Tat with cyclin T1. Flag-tagged Tat was expressed in 293T cells by transfection (lanes 3 and 5). Cells were transfected with non-targeting (lanes 2 and 3) or CDK2-specific (lanes 4 and 5) siRNAs. Lysates were precipitated with anti-Flag antibodies, resolved on 12% Tris-Tricine PAGE and immunoblotted with anti-Tat, anti-CDK9 or anti-CDK2 antibodies. Lane 1, input.

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