Fig. 6: Stub1 promotes TBK1 degradation through the CMA-dependent autophagy pathway. | Cell Death & Differentiation

Fig. 6: Stub1 promotes TBK1 degradation through the CMA-dependent autophagy pathway.

From: E3 ubiquitin ligase Stub1 enhances viral replication by promoting TBK1 degradation through molecular chaperone-mediated autophagy

Fig. 6: Stub1 promotes TBK1 degradation through the CMA-dependent autophagy pathway.

A HEK293T cells were transfected with Flag-TBK1, Myc-Stub1, and HA-HSC70 plasmids for 24 h, and then the lysates were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting. B, C THP-1 cells were infected with VSV or HSV-1 for the indicated times and then subjected to immunoprecipitation with a TBK1 antibody before immunoblot analysis of endogenous TBK1 and HSC70 protein levels. D BIFC analysis of TBK1 and HSC70. HA-HSC70-VN, HSC70 fused with the N-terminus of Venus; Flag-TBK1-VC, TBK1 fused with the C-terminus of Venus. E Co-IP and immunoblot analysis of HEK293T cells transfected with Flag-TBK1 or Flag-TBK1-K344R and HA-HSC70. F HEK293T cells were transfected with a Flag-TBK1 plasmid with or without HA-HSC70 plasmids and then treated with VER-155,008 (5 μM, 10 μM, or 25 μM). TBK1 protein levels were analyzed by immunoblotting. G HEK293T-hsc70−/− cells were transfected with Flag-TBK1, Myc-Stub1, and HA-HSC70 plasmids for 24 h, after which the lysates were analyzed by immunoblotting. H HEK293T-hsc70+/+ and HEK293T-hsc70−/− cells were transfected with Flag-TBK1 and HA-Stub1 plasmids for 24 h, after which the lysates were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting in the presence of CQ (50 μM) for 6 h. I HEK293T-stub1+/+ and HEK293T-stub1−/− cells were transfected with Flag-TBK1 and HA-HSC70 plasmids for 24 h, and then the lysates were immunoprecipitated with an anti-Flag antibody and analyzed by immunoblotting in the presence of CQ (50 μM) for 6 h. J HEK293T-hsc70+/+ and HEK293T-hsc70−/− cells were infected with VSV for 0, 4, 8, or 12 h in the presence of CQ (50 μM). Lysosomes were subsequently isolated and enriched before the proteins were analyzed by immunoblotting. K Fluorescence images showing the colocalization of TBK1 (green) with HSC70 (cyan) on lysosomes (red) in hsc70+/+ cells, whereas TBK1 (green) does not colocalize with lysosomes (red) in hsc70−/− cells; original magnification ×600. The data are representative of three independent experiments with three biological replicates (means ± SDs) or represent three independent experiments with similar results (A–K).

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