The effect of Hsp70 on LPS-induced TNF-α production depends on the binding of HSF-1 and CHBF to the TNF-α promoter. Top panels. Monocytes were stimulated with Hsp70 (3 μg/ml), LPS (100 ng/ml) or Hsp70 + LPS for 15, 30, 60 and 120 min, at 37°C. Non-stimulated cells were used as a negative control (−) and monocytes cultured at 42°C for the indicated times (heat-shock, HS) were used as a positive control. After incubation, nuclear extracts were obtained and analyzed by EMSA, using the TNF-α probe. The autoradiography is representative of 3 independent experiments. The slower migrating complex, corresponds to the binding of HSF-1 to the HSE in the TNF-α promoter, and the faster migrating complex, corresponds to the binding of CHBF to the HSE. Control included EMSA reaction with 50-fold excess cold TNF-α competitor or 50-fold excess irrelevant competitor. Bottom panels. The levels of TNF-α were determined in the supernatant of the same cultures; data is from 3 independent experiments. Two-way ANOVA with Bonferroni post-test: ***P < 0.001.