Fig. 6. CIITA expression in IRF-2 antisense stable transformants of WM9. A, IRF-2 in antisense IRF-2-transformed WM9 cells. A1, A2, A3, and A4 represent four subclones of WM9 transformed with the antisense IRF-2 expression vector. C1 and C2 represent two subclones of WM9 transformed with the empty vector alone and are used as controls. A total of 20 µg of nuclear extract from each subclone was assayed for IRF-2 by immunoblotting. Immunoblotting of ß-actin was used as a loading control. The relative IRF-2 level was determined by normalizing IRF-2 to ß-actin, as quantified by densitometry. B, constitutive and IFN-{gamma}-induced CIITA expression in IRF-2 antisense stable transformants of WM9. Subclones from IRF-2 antisense transformation (A1, A2, and A4) as well as vector transformation (C1 and C2), were either untreated or treated with 100 or 400 IU of rIFN-{gamma} for 48 h for a CIITA RPA. The relative CIITA level was determined by normalizing CIITA mRNA to {gamma}-actin mRNA. Signals obtained from a short exposure of {gamma}-actin RPA gel (middle panel) were used for quantification. Similar results were obtained with A3 (see panel A, above), i.e., A3 had a reduced level of CIITA expression compared with control transformants (data not shown).