FIG. 3.
Characterization of vIL-6-mediated STAT induction in Hep3B cells. (A) Medium containing vIL-6 (from pSVvIL-6-transfected Hep3B cells) or hrIL-6 (500 U/ml) or control medium (fresh or derived from pSG5-transfected Hep3B cells) was applied to serum-starved Hep3B cultures (15 min), and the cells were harvested for the preparation of whole-cell extracts. These extracts were analyzed by Western blot analysis to detect induced (phosphorylated) STAT1 and STAT3 (left and right panels) or total STAT1 (middle panel; stripped, reprobed PY-STAT1 blot). (B) Various dilutions of pSVvIL-6-transfected cell medium were tested for STAT3-inducing activity. Levels of induced STAT3 (top panel) and total STAT3 (bottom panel) in Hep3B cell cultures treated with pSVvIL-6 (1 to 1/16) or pSG5 (0) transfected cell medium were determined by Western analysis. (C) The amount of vIL-6 present in the vIL-6 transfected cell medium was determined by dot blot analysis with a vIL-6 peptide antiserum (see Materials and Methods). The signal from 3 ml of undiluted vIL-6 stock (corresponding to the maximum amount of vIL-6 used for STAT3 induction in panel B) was compared to signals obtained from a dilution series of purified GST–vIL-6 fusion protein to determine the amount of vIL-6 present. The calculated amounts of the vIL-6 component of the GST-vIL-6 fusion protein at each dilution is indicated. Control medium (from pSG5-transfected Hep3B cells) gave no background signal. Also shown are the results of analyses of the expression of vIL-6 variants 15 and 24 (Table 2) secreted from transfected Hep3B cells. (D) The activity and concentration of bacterially synthesized vIL-6 (see Materials and Methods) were determined by STAT3 induction and dot blot assays analogous to those used for Hep3B-expressed vIL-6. Different amounts (in microliters) of bacterial extract (His6vIL-6) were used for each; amounts (in nanograms) of the vIL-6 component of blotted GST–vIL-6 are indicated.