gfp mdc1  (Addgene inc)

Journal: PLOS ONE

Article Title: All- trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication

doi: 10.1371/journal.pone.0305350

Figure Lengend Snippet: (a) HepG2 and Hep3B cells, cultured on coverslips, were transfected with an HBx expression plasmid for 24 h and subsequently either mock-treated or treated with 5 μM ATRA for an additional 24 h. The cells were then processed for double-label indirect immunofluorescence, incubating with anti-HBx monoclonal and anti-E6AP antibodies, followed by incubation with anti-mouse IgG–FITC and anti-rabbit IgG–rhodamine antibodies to visualize HBx (red) and E6AP protein (green), respectively. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). (b to e) Either HepG2 or Hep3B cells were transfected with the indicated plasmids for 24 h and then treated with ATRA at the indicated concentrations for an additional 24 h before harvesting, followed by western blotting. For (d), cells were treated with Heclin at the indicated concentrations for 24 h before harvesting.

Article Snippet: Plasmid cMVT N-HA-hE6AP with human HA-tagged E6AP (amino acids 262–853) and pCH110 which encodes the Escherichia coli β-galactosidase gene were acquired from Addgene (Watertown, MA, USA).

Techniques: Cell Culture, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Incubation, Staining, Western Blot

Journal: PLOS ONE

Article Title: All- trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication

doi: 10.1371/journal.pone.0305350

Figure Lengend Snippet: After 24 hours of transfection with the specified amounts of HBx expression plasmid, both HepG2 and Hep3B cells were treated with ATRA for an additional 24 hours. (a) The levels of the indicated proteins were assessed by western blotting. (b) Methylation-specific PCR (MSP) was conducted to determine the methylation status of CpG sites within the E6AP promoter, distinguishing between unmethylated (U) and methylated (M) states.

Article Snippet: Plasmid cMVT N-HA-hE6AP with human HA-tagged E6AP (amino acids 262–853) and pCH110 which encodes the Escherichia coli β-galactosidase gene were acquired from Addgene (Watertown, MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Methylation

Journal: PLOS ONE

Article Title: All- trans retinoic acid downregulates HBx levels via E6-associated protein-mediated proteasomal degradation to suppress hepatitis B virus replication

doi: 10.1371/journal.pone.0305350

Figure Lengend Snippet: (a) HepG2 and Hep3B cells were transiently transfected with HA-HBx and treated with ATRA as in and then dosed with 50 μM of protein synthesis inhibitor cycloheximide (CHX) for the presented time before harvesting, then followed by western blotting. Using Image J 1.53k image analysis software (NIH), γ-tubulin and HBx levels were quantified. The calculation of half-life (t 1/2 ) of HBx was conducted based on the levels of HBx compared to the loading control (γ-tubulin). (b) HepG2 cells were transiently transfected with the indicated plasmids and then ATRA treatment was conducted as in . Immunoprecipitation was conducted using an anti-HBx antibody to isolate total HBx protein from cell lysates. Subsequently, western blot analysis was performed using suitable antibodies to detect p53, E6AP, Siah-1, HBx, and HA-Ub-complexed HBx. Levels of the specified proteins in the cell lysates are depicted in the input lane. (c) HepG2 cells were transiently transfected with a plasmid containing HBx gene and treated with ATRA as in . (c) Cells prepared following the protocol in were subjected to either mock treatment or treatment with MG132 for 4 h before cell harvest, and subsequent western blotting was performed.

Article Snippet: Plasmid cMVT N-HA-hE6AP with human HA-tagged E6AP (amino acids 262–853) and pCH110 which encodes the Escherichia coli β-galactosidase gene were acquired from Addgene (Watertown, MA, USA).

Techniques: Transfection, Western Blot, Software, Immunoprecipitation, Plasmid Preparation