Fig. 3.

Depletion of HERC5 inhibits the induction of ISG15 conjugation by IFN-β. (A) HERC5 depletion by siRNAs inhibits ISG15 conjugation induced by IFN-β in A549 cells. The A549 cells were transfected with control siRNA (lane 1) or siRNAs against HERC5 (lanes 2 and 3). Twenty-four hours after transfection, A549 cells were treated with IFN-β for another 48 h. Total lysates were harvested and separated in SDS/PAGE. ISG15 conjugates were detected by anti-ISG15 antibody. (B) Suppression of HERC5 mRNA expression in A549 cells by siRNAs. Efficiency of siRNA–HERC5-I and siRNA–HERC5-II in inhibition of HERC5 RNA expression was determined in A549 cells transfected with siRNA control and siRNAs for HERC5 24 h after IFN-β treatment. Expression of HERC5 mRNA was analyzed by real-time PCR with specific primers for HERC5. (C) HERC5 elimination has no effect on the induction of Ube1L and UbcH8 by type I IFN. Total lysates were prepared from A549 cells transfected with control siRNA (lane 1) or siRNAs against HERC5 (lanes 2 and 3) at 24 h after IFN-β treatment. The levels of protein expression of Ube1L and UbcH8 were detected by antibodies against Ube1L (C Upper) and UbcH8 (C Lower), respectively. (D) Suppression of IFN-β-induced HERC5 mRNA expression in HeLa cells constitutively expressing shRNA–HERC5–1-4. Efficiency of shRNA–HERC5–1-4 in inhibition of endogenous HERC5 RNA expression was determined by using real-time PCR with HERC5-specific primers. (E) Inhibition of IFN-β-induced ISG15 conjugation in HeLa cells stably expressing shRNA against HERC5. HeLa cells were stably transfected with vectors expressing shRNA–HERC5–1-4 and selected with 1 μg/ml puromycin. Cells were treated with IFN-β for 48 h, and ISG15 conjugates were analyzed by immunoblotting with anti-ISG15 antibody.