(A) Interaction of MA with IQGAP1 in vitro. Wild-type MA, MA mutants (T4, T5, SMA6, and T11), and MA revertants (T4Rev, T1 Rev, T6Rev1, and T6Rev2) were expressed in bacteria as fusions with the MBP, bound to amylose–sepharose beads, and used as an affinity matrix for the binding of IQGAP1 expressed in bacteria as a fusion to (GST-IQGAP1; aa 1440–1657) or (GST-IQGAP1 Y1627H; aa 1440–1657). The beads were washed and the bound proteins were analyzed by SDS–PAGE and Western blot with anti-MA antisera and GST antibodies. (B) Pr65gag and IQGAP1 associate in vivo. 293T cells were cotransfected with either wild-type proviral DNA (pNCS) or the T4 mutant (pNCS-T4), and with either Flag-tagged full-length IQGAP1 or the C-terminal fragment Flag-IQDN4. Lysates were prepared, the Gag proteins were immunoprecipitated with anti-CA antiserum, and the associated proteins were analyzed by SDS–PAGE and Western blot with anti-FLAG antibodies and anti-CA antisera. As a negative control, samples were also immunoprecipiated with anti-HA antibodies. A portion of the input samples were analyzed by Western blot without immunoprecipitation.