Figure 4.

Effects of CD40 ligation and ectopic expression of RelB on BJAB cell surface markers. (A) BJAB were transfected with 10 μg RelB (dotted line), 10 μg RelB, and 10 μg p50 (bold line) or 10 μg pCDNA3 (thin line). In each case, BJAB were cotransfected with 5 μg pGreenLantern and incubated for 24 h before staining with PE-conjugated anti-CD40 or anti-MHC class I. Cells were analyzed by two-color flow cytometry, gating on GFP⁺ cells. Isotype controls are indicated by the filled histograms. Significant differences were observed in levels of CD40 (see Supplementary Fig. 7A at www.pnas.org) (P = 0.0212) and MHC class I (see Supplementary Fig. 7B at www.pnas.org) (P = 0.0107) after transfection of RelB in BJAB. Data are representative of three separate experiments. (B) Cytoplasmic or nuclear extracts from BJAB transfected with RelB (lanes 3 and 7) or pCDNA3 (lanes 2 and 6) were immunoprecipitated after 24 h with anti-p50, and immunoblots were probed with anti-RelB. p50-blocking peptide was included in the immunoprecipitation of extracts of RelB- (lanes 5 and 9) or pCDNA3- (lanes 4 and 8) transfected BJAB. Lane 1 indicates constitutive RelB present in BJAB cytoplasmic extracts.