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. Author manuscript; available in PMC: 2007 Sep 20.

Published in final edited form as: Mol Immunol. 2006 Jun 12;44(6):1461–1470. doi: 10.1016/j.molimm.2006.04.026

Fig. 3 — Repression of the CIITA type IV promoter by PRDI-BF1 requires sequences within the IRF-E site. (A) Raji B cells were transiently co-transfected by electroporation using 10 μg of the wild-type or mutated CIITA pIV luciferase reporter plasmids and 10 μg of the PRDI-BF1 expression plasmid or the empty vector control. After a 6 h recovery period, half of the cultures were treated with 500 U/ml of recombinant human IFN-γ for 24 h before the cultures were harvested. Luciferase activity was measured and normalized to Renilla luciferase activity. Bars show the S.E.M., n = 3. At least three independent experiments have been performed with similar results. (B) EMSA analysis performed using wild-type CIITA pIV probe, in vitro translated PRDI-BF1 protein and specific cold competitors. The cold competitors include a CIITA pIV oligonucleotide with a mutation that disrupts both GAAAG motifs in the IRF-E site (MUT), and oligonucleotides with mutations that disrupt the first (5′MUT) or second (3′MUT) GAAAG motif used alone or in combination. Incubation with either of the cold competitor oligonucleotides in which a single unmutated GAAAG motif is present is capable of diminishing the formation of the specific complex (arrow), compare lanes 3 and 4 with lanes 1 and 2. No additional reduction is seen when both mutated oligonucleotides were used in combination (compare lane 5 with lanes 3 and 4).

Fig. 3 — Repression of the CIITA type IV promoter by PRDI-BF1 requires sequences within the IRF-E site. (A) Raji B cells were transiently co-transfected by electroporation using 10 μg of the wild-type or mutated CIITA pIV luciferase reporter plasmids and 10 μg of the PRDI-BF1 expression plasmid or the empty vector control. After a 6 h recovery period, half of the cultures were treated with 500 U/ml of recombinant human IFN-γ for 24 h before the cultures were harvested. Luciferase activity was measured and normalized to Renilla luciferase activity. Bars show the S.E.M., n = 3. At least three independent experiments have been performed with similar results. (B) EMSA analysis performed using wild-type CIITA pIV probe, in vitro translated PRDI-BF1 protein and specific cold competitors. The cold competitors include a CIITA pIV oligonucleotide with a mutation that disrupts both GAAAG motifs in the IRF-E site (MUT), and oligonucleotides with mutations that disrupt the first (5′MUT) or second (3′MUT) GAAAG motif used alone or in combination. Incubation with either of the cold competitor oligonucleotides in which a single unmutated GAAAG motif is present is capable of diminishing the formation of the specific complex (arrow), compare lanes 3 and 4 with lanes 1 and 2. No additional reduction is seen when both mutated oligonucleotides were used in combination (compare lane 5 with lanes 3 and 4).