Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Huh-7 cells were transfected with A3G and increasing amounts of HBx as indicated. eGFP expression plasmid was used as transfection control. Cells were harvested 48 hours post transfection and cell lysates were analyzed in parallel in immunoblot. ( B ) Relative quantities of A3G in cell lysates as shown in ( A ) were estimated using densitometry scanning and normalized to eGFP quantities, taking values without HBx co-transfection as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Western Blot, Cotransfection

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Time-course study of A3G protein level following cycloheximide (CHX) treatment. Huh-7 cells transfected with A3G and eGFP expression plasmids, with or without HBx plasmid, were treated with CHX at 48 hours post transfection. Cells were collected at indicated time points and analyzed in parallel using immunoblot. ( B ) Relative quantities of A3G (top) and HBx (bottom) in cell lysates as shown in ( A ) were estimated using densitometry scanning and normalized against eGFP quantities, taking values at start of CHX treatment as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Lack of effects of HBx on A3G ubiquitination as detected using immunoprecipitation. Huh-7 cells transfected with indicated plasmids were treated with MG-132 and subjected to cell lysis and purification of His-tagged A3G using Ni 2+ -NTA matrix under denaturing condition. Enriched His-tagged A3G was detected using anti-A3G and A3G modified by HA-tagged ubiquitin (HA-Ub) was detected using anti-HA antibodies. A3G and HBx in 1/10 input were also detected. ( B and C ) Treatment with proteasome inhibitor MG-132 or lysosome inhibitor leupeptin had no marked effect on A3G protein levels, with or without HBx. Huh-7 cells transfected and treated as indicated were lysed and subjected to immunoblot analysis.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Transfection, Lysis, Purification, Modification, Western Blot

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Detection of A3G protein in cell lysates and exosomes prepared from Huh-7 cells transfected with A3G expression plasmid or vector control. Exosome markers Alix and Rab7, and mitochondria marker VDAC1 were detected in parallel. ( B ) Treatment with GW4869 or transfection of plasmid expressing nSMase2-targeting shRNA (shnSMase2) inhibited exosome secretion by Huh-7 cells. Exosomes were prepared from 1E7 cells treated as indicated and total protein contents were quantified using BCA assay. DMSO vehicle and unrelated shRNA construct (shneg) were used as controls. ( C ) Treatment with GW4869 or shnSMase2 inhibited A3G export via exosome secretion and increased intracellular A3G protein level. Huh-7 cells transfected with A3G expression plasmid or vector control and treated as indicated were used for lysate and exosome preparation. In addition to A3G and exosome/mitochondria markers, nSMase2 was also detected to demonstrate knock-down by shnSMase2.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Marker, shRNA, BIA-KA, Construct, Knockdown

Journal: Scientific Reports

Article Title: Hepatitis B virus X protein is capable of down-regulating protein level of host antiviral protein APOBEC3G

doi: 10.1038/srep40783

Figure Lengend Snippet: ( A ) Effects of HBx on A3G protein levels in cell lysates and exosomes. Huh-7 cells were transfected with indicated plasmids and 48 hours post transfection, cell lysates and exosomes were prepared and analyzed in immunoblot for A3G and HBx levels as well as exosome markers Tsg101 and Rab7. β-actin was used as loading control. ( B ) Relative quantities of A3G and exosome markers Tsg101 and Rab7 in cell lysates and exosomes as shown in ( A ) were respectively estimated using densitometry scanning taking values from vector-transfected cells as 1 (100%). Means and SD calculated from 3 independent experiments are shown.

Article Snippet: A3G (60100-1) and Rab7 (55469-1-AP) antibodies were purchased from Proteintech.

Techniques: Transfection, Western Blot, Control, Plasmid Preparation