Figure 3.

Requirement for proline-rich or serine/threonine-rich domain in addition to the acidic domain for CIITA activity. (A) G3A cells were cotransfected with the pDRA300CAT and pcDNA3 (lane 1), FLAG.CIITA8 (lane 2), or mutant constructs (lanes 3–5). The cells were harvested 48 h after transfection. CAT activity was analyzed and quantitated by PhosphorImager (Molecular Dynamics). The relative level of expression was calculated by using the wild-type control as a value of 1. These experiments were repeated three times. (B) Immunoblot analysis of wild-type FLAG.CIITA8 and mutant constructs. Cells transfected with FLAG.CIITA and mutant CIITA were harvested and lysed 24 h after transfection. Equivalent amount of total lysates from each sample was fractionated on 8% SDS-PAGE. The blot was examined with the α-CIITA antibody and visualized by ECL, according to the manufacturer’s specification. All of the mutant constructs were expressed at a comparable level to the FLAG.CIITA8. No band was observed in cells transfected with the empty vector pcDNA3 (lane 1).