FIG. 4.

Bacterially expressed PACT and PACTΔ3, but not Δ1,2, Δ1, or MBP-3, bind Us11C. GST pulldown assays were used to measure the binding of PACT and PACT mutants to GST-tagged Us11C. One microgram of purified PACT or PACT mutant protein was mixed with 1 μg of purified GST-Us11C in high-salt buffer. Glutathione-Sepharose 4B was used to pull down GST-Us11C, and the proteins interacting with it were analyzed. (A) Purified PACT proteins (1 μg each), representing those that were tested for GST-Us11C binding. Lanes 1 to 4, histidine-tagged PACT proteins Western blotted with anti-histidine antibody; lane 1, wt PACT; lane 2, PACTΔ1; lane 3, PACTΔ1,2; lane 4, PACTΔ3; lane 5, MBP-3 Western blotted with anti-PACT domain 3 antibody. (B to D) Proteins pulled down with glutathione-Sepharose 4B and Western blotted with anti-histidine antibody (B), anti-PACT domain 3 antibody (C), and anti-GST antibody (D). In panels B to D, all lanes except lanes 1 contained GST-Us11C. The additional proteins were as follows: wt PACT, PACTΔ1, PACTΔ1,2, PACTΔ3, and MBP-3 (lanes 1); wt PACT (lanes 2); PACTΔ1 (lanes 3); PACTΔ1,2 (lanes 4); PACTΔ3 (lanes 5); and MBP-3 (lanes 6)