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. 2002 Mar;13(3):915–929. doi: 10.1091/mbc.01-08-0383

Figure 3.

Figure 3

The ZF domain of FBI-1 is necessary and sufficient for Tat association in vivo. (A) Constructs expressing T7FL, T7ΔPOZ, T7ZF, or T7ΔZF and HA-Tat were expressed alone or in combination as indicated above the lanes into HeLa cells. Thirty-six hours posttransfection, extracts from transfected cells were prepared and incubated with protein A-agarose beads cross-linked to anti-HA 12CA5 monoclonal antibodies (lanes 2–9). The immunoprecipitated proteins were fractionated on a 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose and blotted with anti-T7 antibody. Lane 1: one tenth the starting material for the T7FL + HA-Tat immunoprecipitation (IP; lane 6). Lanes 2–9: the IPs. The position of T7FL, T7-ΔPOZ, and T7-ZF are indicated. For lanes 6–9, an anti-HA immunoblot of a separate gel demonstrated that the HA-tagged proteins were expressed and immunoprecipitated (our unpublished results). For lanes 6–9, an anti-T7 immunoblot of a separate gel loaded with one tenth the starting material for the IP demonstrated that the mutant proteins were expressed to a similar level (B). (C) Constructs expressing T7ZF and wild-type HA-Tat, HA-18IS Tat, or HA-C30, 31A Tat were transfected alone or in combination as indicated above the lanes into HeLa cells. The experiment was performed as in (A). Lanes 1–4 show one tenth the starting materials; lanes 5–8 show the IPs.