FIG. 6.
Phosphorylation of A20 S381 leads to rapid inhibition of NF-κB following LPS stimulation. (A) A20−/− MEFs were stably reconstituted with Flag-A20 WT, Flag-A20 S381A, or vector control and pulsed with 2 ng/ml TNF-α for 30 min. After extensive PBS washes, the TNF-α-containing medium was replaced with serum-free medium and the recovery of IκBα levels was observed over time via immunoblotting of lysates. (B) Reconstituted A20−/− MEFs were pulsed with 2 ng/ml TNF-α for 30 min, the medium was replaced as described above, and cells were allowed to recover for 15 min. Lysates were immunoblotted using an anti-IκBα antibody, and differences were calculated between IκBα levels in WT A20-reconstituted versus A20 S381A- or vector-reconstituted MEFs. The experiment was repeated four times with similar results. Error bars show standard deviations. (C) Stably reconstituted A20−/− MEFs were pulsed with a higher dose (6 ng/ml) of TNF for 30 minutes. After extensive PBS washes, the TNF-containing medium was replaced with serum-free medium and the recovery of IκBα levels was assayed via Western blotting.