retrograde aav1 syn jgcamp8s wpre  (Addgene inc)

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core stimulates E6AP-mediated proteasomal degradation of p53 but inhibits MDM2-mediated degradation. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the indicated plasmids, for 48 h. For ( a ), HepG2-Core cells stably expressing HCV Core were also included. ( a , b ) Protein levels were measured by Western blot analysis. ( c ) Cells were treated with 50 μM cycloheximide (CHX) for the indicated times before harvesting, followed by Western blotting. Bands of p53 and γ-tubulin were quantified to determine the half-life (t1/2) of p53. The difference in the p53-to-γ-tubulin ratio among samples is shown in a graph. ( d , e ) Cells were treated with 10 μM MG132 for 4 h before harvesting to block further proteasomal degradation. For lanes 5 and 6 in ( e ), cells were treated with Nutlin 3a for 24 h. Total p53 was immunoprecipitated with an anti-p53 antibody and subjected to Western blotting using anti-E6AP, anti-MDM2, anti-HCV Core, and anti-HA antibodies to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed p53, respectively. The input indicates the levels of the designated proteins in the cell lysates. ( f , g ) For mammalian two-hybrid assays, Hep3B cells were transfected with the Gal4 reporter (G5E1b-luc), pSG424-E6AP (or pSG424-MDM2), and pCMV p53-VP16, along with the indicated plasmids, for 48 h, followed by a luciferase assay. For ( f ), cells were treated with 10 μM MG132 for 4 h before harvesting. Luciferase activity from G5E1b-luc was normalized to the β-gal activity measured in the corresponding cell extract. The values show relative luciferase activity compared to the control’s basal level. Results are presented as mean ± SD from four independent experiments ( n = 4). ND, not detected.

Article Snippet: Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [ ], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA).

Techniques: Transfection, Plasmid Preparation, Expressing, Stable Transfection, Western Blot, Blocking Assay, Immunoprecipitation, Luciferase, Activity Assay

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: E6AP induces ubiquitin-dependent proteasomal degradation of phosphorylated p53 in the presence of HCV Core. HepG2 and Hep3B cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated plasmids, for 48 h. For ( c , d ), cells were treated with 10 μM MG132 for 4 h before harvesting. Band intensities were quantified using ImageJ (NIH). ( a , d ) Protein levels were measured by Western blot analysis. ( b ) Phosphorylated p53 at Ser-15 (pSer-15 p53) in cell lysates was immunoprecipitated with an anti-pSer-15 p53 antibody, followed by Western blotting to detect E6AP, MDM2, HCV Core, and HA-Ub-complexed pSer-15 p53. The input shows the levels of the indicated proteins in cell lysates. ( c ) E6AP or MDM2 were immunoprecipitated with the appropriate antibodies and subjected to Western blotting to detect HCV Core, p53, pSer-15 p53, and pSer-20 p53. The input shows the levels of the indicated proteins in cell lysates. ND, not detected.

Article Snippet: Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [ ], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core-induced p53 phosphorylation is required for E6AP-mediated proteasomal degradation. ( a ) HepG2 cells transiently transfected with the designated plasmids for 47 h were treated with the indicated concentrations of the ATM inhibitor KU-55933 for 1 h before harvesting, followed by Western blotting. ( b , c ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53 or anti-E6AP antibodies, followed by Western blotting. ( d – f ) For mammalian two-hybrid assays, Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 47 h. Cells were either mock-treated or treated with 10 μM KU-55933 for 1 h before harvesting, followed by a luciferase assay ( n = 4). ND, not detected.

Article Snippet: Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [ ], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA).

Techniques: Phospho-proteomics, Transfection, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: p53 phosphorylation is sufficient for E6AP to induce p53 ubiquitination. ( a ) HepG2 cells were transiently transfected with either an empty vector or the HCV Core expression plasmid, along with the designated amounts of the E6AP expression plasmid, for 48 h. Cells were treated with the designated concentrations of etoposide for 24 h and MG132 for 4 h before harvesting, followed by Western blotting. ( b – d ) Cell lysates prepared in ( a ) were immunoprecipitated with anti-p53, anti-pSer-15 p53, or anti-E6AP antibodies, followed by Western blotting. ( e – g ) Hep3B cells were transfected with pSG424-E6AP (or pSG424-MDM2), pCMV p53-VP16, and G5E1b-luc, along with the indicated plasmids, for 48 h. Cells were either mock-treated or treated with 10 μM etoposide for 24 h before harvesting, followed by a luciferase assay ( n = 4).

Article Snippet: Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [ ], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Expressing, Western Blot, Immunoprecipitation, Luciferase

Journal: Cells

Article Title: Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

doi: 10.3390/cells15050415

Figure Lengend Snippet: HCV Core triggers E6AP-dependent ubiquitination of p53 during HCV replication in Huh7D cells. ( a – d ) Huh7D cells were transfected with specific plasmids for 24 h, then infected with HCV for another 24 h. ( a – c ) Protein levels were detected by Western blotting. Myc-p53 indicates p53 from ectopic expression, while Y220C-p53 represents the endogenous form in Huh7D cells. For ( b ), cells were either mock-treated or exposed to the indicated concentrations of Heclin for 12 h before harvesting. ( d ) Cell lysates were immunoprecipitated with an anti-Myc antibody, and the precipitates were analyzed by Western blotting. ( e ) Schematic diagram illustrating how HCV Core regulates proteasomal degradation of p53 mediated by MDM2 and E6AP. Each step is described in the Discussion.

Article Snippet: Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [ ], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA).

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