In vivo and vitro interactions of the EICE and E-boxes of CIITA-PIII in B cells. (A) ChIP analysis of CIITA-PIII and CIITA-PIV in Raji B cells with antisera specific for E47 (lane 2), PU.1 (lane 3), STAT-1 (lane 4), IRF-4 (lane 11), IRF-8 (lane 12), and ac-H3 (lane 13). PCR products specific for both CIITA-PIII (top panels) or CIITA-PIV (bottom panels) are indicated. PCR products obtained with input DNA are shown in lanes 6 and 9, and PCR products obtained without the addition of a primary antibody are shown in lanes 5 and 10. The 1-kb marker and the negative water control are indicated in lanes 1, 7, and 8, respectively. (B) EMSA with NE derived from Raji B cells (lanes 1-6) and splenic B cells (lanes 7-12). EICE-interacting factors were visualized using the CIITA-derived PIII-EICE as a probe and compared to the pattern obtained with a PIII-mut-EICE encoding probe. Supershift antibodies and competition probe are indicated on the top of each plot, and supershifts were performed with antibodies specific for PU.1 (lanes 3 and 9), IRF-4 (lanes 4 and 10), IRF-8 (lanes 5 and 11), and STAT-1 (lane 13). Competition was performed using a 3′Eλ-EICE probe (lanes 2 and 8) that encodes the consensus EICE sequence of the 3′Eλ_2-4 and the PIII-CRE (ARE-2 motif) probe (lane 12), as described previously.^19,21  Supershifted complexes are indicated with an asterisk, and complexes reduced or eliminated after incubation with the PU.1 are indicated by arrows I-IV, of which complexes II and III are also reduced after incubation with IRF-4/8 specific antibodies. (C) EMSA analysis for E-box-interacting factors were performed using a consensus E-box (cons-E-box) and both E-box motifs of CIITA-PIII (473-E-box and 298-E-box) as probes with NE extracts derived from Ramos B cells and Raji B cells. Supershift analysis with E47-specific antibody is indicated on the top of the plots, and E47 complex is indicated with an arrow. Probes are indicated below all EMSA plots.