Figure 5.

FBXW8 induces the autophagic degradation of PDCoV N protein via NDP52. (A) The FBXW8 and PDCoV N plasmid co-transfected cells were exposed to the treatment with ubiquitination inhibitor MG132, autophagy inhibitors 3-MA and CQ, and caspase inhibitor Z-VAD-FMK. (B) HEK-293T cells were transfected with HA-PDCoV-N and increasing concentrations of Flag-FBXW8 for 24h. The WCLs were analyzed with Western blotting. (C) The Flag-tagged cargo receptors (SQSTM1/p62, NDP52, OPTN, BNIP3L, and TOLLIP) were co-transfected with HA-PDCoV-N plasmids into HEK-293T cells, respectively. (D) HEK-293T cells were co-transfected Flag-NDP52 with HA-PDCoV-N plasmids, and the WCLs were purified by anti-Flag or anti-HA affinity gel for immunoblot analysis. (E) The pCold-GST-NDP52 plasmids were transformed into BL21(DE3) cells to induce protein expression. The relationship between NDP52 and PDCoV N protein was assessed with the use of the GST pull-down kit. (F) PK-15 cells were used for expression of Flag-NDP52 (green) and HA-PDCoV N protein (red). Cell nuclei were stained with DAPI (blue). Confocal immunofluorescence microscopy was used to visualize the results. (G, H) HA-FBXW8 and Flag-NDP52 together with Myc-PDCoV-N or empty plasmids were co-expressed in 293T cells, followed by a Co-IP assay utilizing Anti-Flag or Anti-HA affinity gels, respectively. (I) HEK-293T cells were co-transfected Flag-PDCoV N, Myc-FBXW8 with NDP52 shRNA or control (shNC). (J) HEK-293T cells were co-transfected Myc-PDCoV-N, HA-FBXW8 with NDP52 shRNA or shNC. The abundance of specific protein was analyzed by Western blot analysis.