Fig. 6.

IFN-γ-mediated-STAT1 binding to the TAP1 promoter and APM protein expression is independent of STAT1:STAT3 heterodimerization. a PCI-13 and b SCC90 cells were untreated, treated with IL-6 (50 ng/ml, 60 min), IFN-γ (1,000 U/ml, 30 min) or pretreated with IL-6 (50 ng/ml, 30 min), then treated with IFN-γ (1,000 U/ml, 30 additional min) in the presence of IL-6. The cells were fixed with formaldehyde, quenched with glycine and lysed in SDS lysis buffer. Chromatin was sheared by sonication and probed with anti-STAT1, anti-STAT3 and anti-IgG mAbs. Protein–DNA crosslinks were reversed, and both RNA and protein were removed by enzymatic digestion. DNA was purified, and PCR was performed amplifying a canonical GAS sequence in the TAP1 promoter localized to STAT1 binding. c PCI-13 and d SCC90 cell were untreated, treated with IL-6 (50 ng/ml, 48 h), IFN-γ (100 U/ml, 48 h) or pretreated with IL-6 (50 ng/ml, 30 min), then treated with IFN-γ (100 U/ml, 48 h) in the presence of IL-6. Intracellular flow cytometry was performed measuring TAP1, TAP2, LMP2 and calreticulin. MFI was plotted representing at least three independent experiments (P = NS, two-tailed t test). Error bars indicate standard error