Fig 3.

GM-CSF- and TNF-α-treated HPCs have higher nuclear NF-κB DNA binding activity than STIF medium-treated cells. (A) Quantitation of the results of a transcription factor ELISA measuring NF-κB DNA-binding activity in nuclear lysates from HPCs incubated with STIF medium or with GM-CSF plus TNF-α. Absorbance was normalized to that of the positive control on each plate; then nonspecific activity (defined by the activity obtained using cytoplasmic extracts) was subtracted from the nuclear absorbance, and the difference was graphed as arbitrary units (AU). The value for the positive control, a Raji nuclear cell extract, was set to 1 AU. Means and standard errors for 3 independent experiments are shown. Asterisk indicates a significant difference (*, P < 0.05) by a paired t test. (B) Quantitation of the results of a transcription factor ELISA measuring AP-1 DNA-binding activity in nuclear lysates from HPCs incubated with STIF medium or with GM-CSF plus TNF-α overnight. Data were analyzed as described for panel A except that nuclear extracts of K-562 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate were used as the positive control. Means and standard errors for 2 independent experiments are shown. AP-1 nuclear activity was not significantly elevated by GM-CSF and TNF-α treatment (P, >0.2 for all subunits by a paired t test); however, JunB expression in STIF medium-treated cells (P < 0.05) and FosB (P < 0.01) and Fra-1 (P < 0.05) expression in GM-CSF- and TNF-α-treated cells were significantly different from zero by a 1-sample t test.