FIGURE 9.

A20 knockdown abolishes LPS tolerance induction in THP1 cells. THP1 cells were transduced with lentiviral particles encoding copGFP, control shRNA, or A20 shRNA and stable cell pools were selected with puromycin. Thereafter, the cells were pretreated for 20 h with medium or 10 ng/ml LPS, washed, and exposed to medium or 100 ng/ml LPS for the indicated time points (A) or treated for 24 with medium, 100 ng/ml LPS or 100 ng/ml PMA (C). A, protein levels of A20 and tubulin were determined by immunoblot analyses of whole cell lysates with the respective Abs. Similar results were obtained in other two independent experiments. B, densitometric quantification of A20 protein levels normalized to tubulin levels. C, supernatants were collected, and the levels of secreted TNF-α were analyzed by ELISA in triplicate samples. The data of a representative experiment (means ± S.D.) of a total of three experiments are shown.