Cellular extracts (3 mg) from untreated NHDF (□, dashed gray line) and NHDF following treatment with 4 ng/ml TNF for 18h (■, solid black line) were separated by isoelectric focusing in solution. Ampholytes were removed and equivalent amounts of each fraction were assayed for protective activity. Percent protection was calculated as described for Fig. 1A. A570 for untreated NHDF was the same as that for the three controls: CHX alone (Fig 4), fraction alone (data not shown) and fraction + CHX (data not shown). The average percent protection (□,■, n=2-4) is shown. The average pH (of the same fraction number from treated and untreated cellular extracts) for each fraction is shown (▲, dashed black line). Data shown are representative of multiple (>10) experiments using multiple (>10) independently-generated cellular extracts.