CIITA(408–570) is sufficient for self-association. (A) Diagram depicting CIITA mutants used to study interactions. (B) The acidic domain interacts with CIITA(408–857). HA-tagged CIITA(1–331), CIITA(408–857), or CIITA(980–1103) was cotransfected with the FLAG-tagged constructs shown. Anti-HA immunoprecipitation reactions were loaded on SDS–9% PAGE gels and probed with anti-FLAG antibodies (top panel). The blot was stripped and reprobed with anti-HA antibodies (middle panel). One-twentieth of each lysate was analyzed for expression of the FLAG-tagged proteins (bottom panel). HC and LC denote the antibody heavy chain and light chain, and the arrowheads indicate the CIITA mutant proteins for this and subsequent parts of the figure. CIITA(408–570) interacts with itself (C) and the LRR (D). Transfections were done as in previous figures, and 1/20 of the cell lysate (L) or the immunoprecipitation reaction using anti-HA antibodies (IP) was subjected to SDS–10 to 12% to PAGE, followed by Western blot analysis with anti-FLAG antibodies. Each part of the figure is representative of at least three separate experiments.