Endogenous ubiquitin regulates the inhibitory activity for TNF-α production in trauma patients' serum.

(A) Trauma patients' serum was applied to an antiubiquitin antibody column and the adsorbed protein was eluted by acidification. Run-through and elutions were collected and tested for inhibitory activity of LPS-induced TNF-α production in healthy donors' whole blood. Whole blood cultures were incubated with the fractions (50%, vol/vol, in the cell culture mixtures) obtained by affinity chromatography in the presence of LPS for 4 hours in a constant volume of 200 μL. Data (percent control) are means ± SD of the TNF-α secretion in the cell culture supernatants from 2 experiments (in duplicates). C indicates control cell culture in the presence of 25% additional volunteers' serum in a constant volume of 200 μL; TS, cell culture in the presence of 25% trauma patients' serum in a constant volume of 200 μL; RT, cell cultures containing the run-through fraction. pH 7 to pH 3, cell cultures containing the eluted fractions. (B) Immunoblot analysis of the fractions obtained by antiubiquitin affinity chromatography. Fractions were separated by SDS-PAGE, transferred to PVDF membranes, and probed for ubiquitin with antiubiquitin AS (1:200; lanes 1-4) and monoclonal UbP4D1 (1:500; lanes 5-8). Lane 1, patients' serum, 10 μg; lane 2, run-through, 20 μg; lane 3, pH 3/4 eluate, 20 μL; lane 4, ubiquitin, 10 ng; lane 5, patients' serum, 50 μg; lane 6, run-through, 50 μg; lane 7, pH3/4 eluate, 200 μL, pH3/4 eluate 10-fold concentrated by boiling; lane 8, ubiquitin 80 ng.