Figure 3.

Src family kinases activation is essential for interferon regulatory factor 1 (IRF-1) K63-linked ubiquitination. (A) Immunoblot analysis of IRF-1 and pSFKs in hTLR7-HEK293 cells pretreated or not with PP2 (20 µM) for 30 min and stimulated with R848 (10 µM) for 2 h. After stripping, membranes were blotted with anti-actin antibody as loading control. The histograms on the right of the blots show the results of the densitometric analysis on three independent experiments. Each signal was normalized to its respective actin signal on the same blot and expressed as fold induction (mean ± SD) compared to untreated sample. (B,C) hTLR7-HEK293 cells transiently transfected with plasmids coding for hemagglutinin (HA)-tagged K63-only (B) or K48-only ubiquitin mutants (C) were pretreated or not with PP2 (20 µM) for 30 min and stimulated for 2 h with R848 (10 µM). Total cell lysates were immunoprecipitated using an anti-IRF-1 antibody, separated by SDS-PAGE, and immunoblotted using anti-HA antibody for ubiquitin detection. Membranes were stripped and re-probed with an anti-IRF-1 antibody as control (bottom panels). The histograms on the right of the blots show the results of the densitometric analysis on three independent experiments. Each signal was normalized to the signal of the protein used for the pull-down and expressed as fold induction (mean ± SD) compared to untreated sample. ***P < 0.001, **P < 0.01, *P < 0.05.