FIG 6.

STING undergoes proteasome degradation. (A) HFFs were infected with wild-type, v65Rev, or v65Stop virus at an MOI of 1. Lysates were prepared at the indicated time points and subjected to Western blot analysis for pp65, cGAS, STING, and α-tubulin. (B) Western blot analysis for STING in cells transfected with poly(dA-dT) (4 μg/ml) at the indicated time points. Lysates were also stained for α-tubulin as a loading control. (C) HFFs infected with wild-type, v65Rev, and v65Stop viruses (MOI = 1) were treated with MG132 or DMSO. Cells were harvested at 6 and 24 hpi and processed for Western blot analyses with antibodies against STING. Lysates were also stained for pp65 and with α-tubulin as a loading control. (D) Coimmunoprecipitation from virus-infected or mock-infected cell lysates. HFFs were infected with wild-type, v65Rev, or v65Stop virus (MOI = 1) and harvested at 2 hpi. At the indicated time points, total cell protein extracts were immunoprecipitated for ubiquitin and stained with anti-STING antibodies. Immunoprecipitation of ubiquitin-conjugated proteins was performed using a UbiQapture-Q kit (Enzo Life Science). (E) Cells were infected as described for panel D. Immunoprecipitations were performed using ubiquitin-K48-specific antibodies. The immunoprecipitated proteins were detected by Western blot analyses using antibodies against STING. Nonimmunoprecipitated whole-cell extracts (Input) were immunoblotted using anti-STING and with α-tubulin antibody as a loading control.