Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: The ubiquitin-proteasome pathway plays a major role in the degradation of pCH25H during PRRSV infection. (A) PAMs were infected with PRRSV (MOI of 1.0). At various time points (24, 36, 48, and 60 h) postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. (B to F) PAMs were infected with PRRSV (MOI of 1.0) and treated with various inhibitors, including MG132 (10 μM) (B), NH4Cl (10 mM) (C), Z-VAD-FMK (10 μM) (D), CQ (20 μM) (E), or 3-MA (5 mM) (F). At 24, 36, and 48 h postinfection, cells were harvested and pCH25H expression was analyzed by Western blotting. ImageJ software was used to analyze the relative levels of pCH25H in comparison with mock-infected cells, and the ratios are displayed as fold changes below the images. DMSO, dimethyl sulfoxide.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Ubiquitin Proteomics, Infection, Expressing, Western Blot, Software, Comparison

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: PRRSV E protein degrades pCH25H via the ubiquitin-proteasome pathway. (A) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. At 24 h posttransfection, cells were treated for 24 h with MG132 (10 μM), Z-VAD-FMK (10 μM), 3-MA (5 mM), or NH4Cl (10 mM). Cell lysates were harvested and analyzed by Western blotting for analysis of expression of PRRSV E protein and pCH25H. (B) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. Cells were treated with different concentrations of MG132 at 24 h posttransfection. Cell lysates were harvested and analyzed by Western blotting for analysis of pCH25H expression. (C and D) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged E protein and FLAG-tagged pCH25H. Cells were lysed at 48 h posttransfection and immunoprecipitated with anti-FLAG antibody. The whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-β-actin, and anti-ubiquitin (Ub) (C) or anti-K48 (D) antibodies. DMSO, dimethyl sulfoxide.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Virology

Article Title: Porcine Reproductive and Respiratory Syndrome Virus E Protein Degrades Porcine Cholesterol 25-Hydroxylase via the Ubiquitin-Proteasome Pathway

doi: 10.1128/JVI.00767-19

Figure Lengend Snippet: The Lys28 site of pCH25H is ubiquitinated by PRRSV E protein. (A) CH25H sequences from different species were aligned (*, lysine). (B) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged wild-type (WT) pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A) and HA-tagged PRRSV E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. The numbers below the images represent the relative levels of pCH25H, compared to that of the corresponding control group, as determined via ImageJ analysis. (C) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged PRRSV E protein and FLAG-tagged wild-type pCH25H or pCH25H mutants (K28A, K72A, K148A, K158A, K259A, or K270A). The cells were lysed at 48 h after transfection and immunoprecipitated with an anti-FLAG antibody. Whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-ubiquitin (Ub), or anti-β-actin antibodies. (D) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged K28A and different amounts of expression vectors encoding HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting. (E) 3D4/21 cells were cotransfected with expression vectors encoding FLAG-tagged pCH25H or hCH25H and HA-tagged E protein. At 48 h posttransfection, cells were harvested for analysis of pCH25H expression by Western blotting.

Article Snippet: Rabbit polyclonal antibodies against ubiquitin and K48-ubiquitin were purchased from Abclonal (Wuhan, China).

Techniques: Expressing, Western Blot, Control, Transfection, Immunoprecipitation, Ubiquitin Proteomics