HeLa cells were cultured with medium containing penicillin/streptomycin and 10% FBS at 37°C in the presence of 5% CO2. Cells were
stimulated with 100 ng/mL human IFN-β for 48 h, and then incubated with 10 µM MG132 (Sigma) for 4 h before harvest. HeLa cells were treated with 10 µM MG132 for 30 min and then stimulated with 10 ng/mL TNF-α for 15 min at 37°C. Cells were harvested and treated as previously described [46 (link)]. The 20 µL reaction volumes contained 200 nM DUBs and 10 µg of total lysate with 25 mM DTT; reactions were terminated by heating with SDS loading buffer and analysed using SDS-PAGE and western blotting with the indicated antibodies. Antibodies used in this study were anti-ubiquitin (P4D1, Santa Cruz Biotech), anti-ISG15 (Bioswamp), anti-His (Proteintech), anti-K48-ubiquitin (Boster), goat anti-rabbit secondary antibodies (Proteintech), and goat anti-mouse IgG light-chain secondary antibodies (Proteintech). Images were captured with an Amersham Imager 600 (GE Healthcare) imaging system.
stimulated with 100 ng/mL human IFN-β for 48 h, and then incubated with 10 µM MG132 (Sigma) for 4 h before harvest. HeLa cells were treated with 10 µM MG132 for 30 min and then stimulated with 10 ng/mL TNF-α for 15 min at 37°C. Cells were harvested and treated as previously described [46 (link)]. The 20 µL reaction volumes contained 200 nM DUBs and 10 µg of total lysate with 25 mM DTT; reactions were terminated by heating with SDS loading buffer and analysed using SDS-PAGE and western blotting with the indicated antibodies. Antibodies used in this study were anti-ubiquitin (P4D1, Santa Cruz Biotech), anti-ISG15 (Bioswamp), anti-His (Proteintech), anti-K48-ubiquitin (Boster), goat anti-rabbit secondary antibodies (Proteintech), and goat anti-mouse IgG light-chain secondary antibodies (Proteintech). Images were captured with an Amersham Imager 600 (GE Healthcare) imaging system.
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