Figure 4.

Recruitment of endogenous and rICSBP to the GAS. (a) Diagram of DNA affinity binding assay. GAS-conjugated beads were incubated with nuclear extracts (NE) with or without rICSBP, and bound materials were detected in immunoblot assays. (b) Binding of endogenous ICSBP to the GAS element: competition analysis. GAS-conjugated beads were incubated with nuclear extracts from RAW cells treated with IFNγ in the absence (lane 1) or presence (lanes 2–5) of a 100- or 25-fold molar excess of WT-GAS or mt-GAS oligomers. Bound (lanes 1–5) or unbound (lane 6, WT GAS competitor; lane 7, mt GAS competitor) fractions were analyzed for ICSBP and STAT1 proteins. (c) Binding assays were performed by using rICSBP or rIRF-1 in the absence (lanes 1–3) or presence of extracts from RAW cells treated without (lanes 5 and 6) or with (lanes 8 and 9) rIFNγ. (d) rICSBP binding in the presence of extracts from CL-2 cells treated without (lanes 1 and 2) or with (lanes 3 and 4) IFNγ. (e) A model for IFNγ action. IFNγ-responsive genes including ICSBP are first activated by the classic JAK/STAT pathway through GAS. ICSBP is recruited to GAS through protein–protein interaction, providing a second wave of transcription from certain IFNγ-inducible genes in an immune-cell-specific manner.