gst bag3 ww proteins  (ProSci Incorporated)

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Schematic diagram of the “proline-rich” reading array chip. Each lettered square contains 12 numbered WW- and/or SH3 GST fusion domains in duplicate. A mock (M) GST sample is in the center of each square. B) The fluorescent pattern following binding of the EBOV VP40-WT biotinylated peptide to the array. The fluorescent spots indicate a positive peptide/WW-domain interaction. EBOV VP40 peptide interactions with WW1 of Rsp5 (square A, green boxes), WW3 of Nedd4 (square B, purple boxes), WW1 of ITCH (square C, yellow boxes), and the BAG3 WW domain (square G, red oval) are highlighted.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Binding Assay

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Extracts from HEK293T cells transfected with eVP40 or eVP40-ΔPT/PY plus BAG3-WT were first immunoprecipitated (IP) with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using mouse anti- myc antiserum. Expression controls for eVP40-WT, eVP40-ΔPT/PY, His-myc-tagged BAG3 and GAPDH are shown. B) Extracts from HEK293T cells transfected with Flag-tagged mVP40 or mVP40(P>A) plus BAG3-WT were first immunoprecipitated (IP) with either normal mouse IgG or anti-Flag antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using rabbit anti-His antiserum. Expression controls for mVP40, mVP40(P>A), His-myc-tagged BAG3 and β-actin are shown. C) Extracts from HEK293T cells transfected with eVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for eVP40-WT, endogenous BAG3, and GAPDH are shown. D) Extracts from HEK293T cells transfected with mVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or anti-mVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for mVP40-WT, endogenous BAG3, and β-actin are shown.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Schematic diagram of BAG3-WT and mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains including the single N-terminal WW-domain (blue), two IPV domains (orange), the PxxP region (yellow), and the BAG domain (green). All three proteins contain both His and cmyc epitope tags. B) Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated (IP) with either rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot (WB) using mouse anti-c myc antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown. C) Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with either mouse IgG or anti-flag (mVP40) antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot using polyclonal anti-His antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were transfected with a constant amount of eVP40 plus vector (-), or increasing amounts of BAG3-WT (A) , BAG3-ΔC (B) , or BAG3-ΔN (C) . The indicated proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control. HEK293T cells were transfected with a constant amount of mVP40 plus vector (-), or increasing amounts of BAG3-WT (D) , BAG3-ΔC (E) , or BAG3-ΔN (F) . The indicated proteins were detected in cell extracts and VLPs by Western blotting. mVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) HEK293T cells were transfected with eVP40 plus either random (control) or BAG3-specific siRNA as indicated. Proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLPs from control cells (lane 1) was set at 1.0. B) Quantification of the relative budding ratio of eVP40 VLPs from four independent experiments. Statistical significance was analyzed by a student t test, *** = p<0.001.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) HEK293T cells were transfected with GFP-eVP40 (green) plus either vector, or BAG3-mCherry (red), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with arrows highlighting the typical localization pattern of GFP-eVP40 at the plasma membrane and in PM projections, while arrowheads highlight the altered diffuse cytoplasmic localization pattern of eVP40 observed in BAG3 expressing cells. Cell nuclei were stained with NucBlue. Scale bars = 10μm. B) HeLa cells were transfected with GFP-eVP40 (green) plus mCherry-LC3 (red) and vector alone (top row), or BAG3 (bottom two rows), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with white arrows highlighting the colocalization of GFP-eVP40 and mCherry-LC3 in aggresomes. Cell nuclei were stained with NucBlue. Scale bars = 10μm.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Plasmid Preparation, Microscopy, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were mock-transfected or transfected with eVP40 (A) or mVP40 (C) plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 (B) or mVP40 (D) from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. E) HEK293T cells were transfected with eVP40 plus vector, BAG3-WT, or BAG3-ΔN as indicated. Cells were fixed at 24 hrs post-transfection, and then incubated with rabbit anti-eVP40 antiserum and mouse anti-myc antiserum (to detecting BAG3-WT and BAG3-ΔN). Cells were then stained with Alexa Fluor 488 goat anti-rabbit and 594 goat anti-mouse secondary antibodies. Microscopy was performed using a Leica SP5 FLIM inverted confocal microscope and XZY scanning. Representative images displaying eVP40 (green) and BAG3-WT (red) or BAG3-ΔN (red) localized at the PM are shown. Cell nuclei were stained with NucBlue. Scale bars = 10μm.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Isolation, Western Blot, Plasmid Preparation, Incubation, Staining, Microscopy

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were first transfected with vector alone, BAG3-WT or BAG3-ΔN for 24 hours, and then infected with recombinant virus VSV-M40 (A) or VSV-M40-P2728A (C) at a MOI of 0.1 for 8 hours. Supernatants were harvested and virus titers were determined by standard plaque assay on BHK-21 cells. Each bar represents the average of three independent experiments performed in duplicate. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. The indicated proteins from VSV-M40 (B) or VSV-M40(P2728A) (D) infected cell extracts were detected by Western blotting.

Article Snippet: GST alone and GST-BAG3 WW domain fusion protein were expressed in BL-21 cells and subsequently conjugated to glutathione (GSH) beads (GE HEALTHCARE).

Techniques: Transfection, Plasmid Preparation, Infection, Recombinant, Plaque Assay, Western Blot

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Schematic diagram of the “proline-rich” reading array chip. Each lettered square contains 12 numbered WW- and/or SH3 GST fusion domains in duplicate. A mock (M) GST sample is in the center of each square. B) The fluorescent pattern following binding of the EBOV VP40-WT biotinylated peptide to the array. The fluorescent spots indicate a positive peptide/WW-domain interaction. EBOV VP40 peptide interactions with WW1 of Rsp5 (square A, green boxes), WW3 of Nedd4 (square B, purple boxes), WW1 of ITCH (square C, yellow boxes), and the BAG3 WW domain (square G, red oval) are highlighted.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Binding Assay

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Extracts from HEK293T cells transfected with eVP40 or eVP40-ΔPT/PY plus BAG3-WT were first immunoprecipitated (IP) with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using mouse anti- myc antiserum. Expression controls for eVP40-WT, eVP40-ΔPT/PY, His-myc-tagged BAG3 and GAPDH are shown. B) Extracts from HEK293T cells transfected with Flag-tagged mVP40 or mVP40(P>A) plus BAG3-WT were first immunoprecipitated (IP) with either normal mouse IgG or anti-Flag antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using rabbit anti-His antiserum. Expression controls for mVP40, mVP40(P>A), His-myc-tagged BAG3 and β-actin are shown. C) Extracts from HEK293T cells transfected with eVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for eVP40-WT, endogenous BAG3, and GAPDH are shown. D) Extracts from HEK293T cells transfected with mVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or anti-mVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for mVP40-WT, endogenous BAG3, and β-actin are shown.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) Schematic diagram of BAG3-WT and mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains including the single N-terminal WW-domain (blue), two IPV domains (orange), the PxxP region (yellow), and the BAG domain (green). All three proteins contain both His and cmyc epitope tags. B) Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated (IP) with either rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot (WB) using mouse anti-c myc antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown. C) Extracts from HEK293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with either mouse IgG or anti-flag (mVP40) antisera as indicated. BAG3-WT or mutant proteins were detected in the precipitates by Western blot using polyclonal anti-His antiserum. BAG3-WT (lane 4) and BAG3-ΔC (lane 6) are indicated by an arrow. Expression controls for the indicated proteins are shown.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Functional Assay, Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Western Blot, Expressing

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were transfected with a constant amount of eVP40 plus vector (-), or increasing amounts of BAG3-WT (A) , BAG3-ΔC (B) , or BAG3-ΔN (C) . The indicated proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control. HEK293T cells were transfected with a constant amount of mVP40 plus vector (-), or increasing amounts of BAG3-WT (D) , BAG3-ΔC (E) , or BAG3-ΔN (F) . The indicated proteins were detected in cell extracts and VLPs by Western blotting. mVP40 VLP production from control cells (lane 1) was set at 100%, and the numbers in () represent relative VLP budding compared to the control.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) HEK293T cells were transfected with eVP40 plus either random (control) or BAG3-specific siRNA as indicated. Proteins were detected in cell extracts and VLPs by Western blotting. eVP40 VLPs from control cells (lane 1) was set at 1.0. B) Quantification of the relative budding ratio of eVP40 VLPs from four independent experiments. Statistical significance was analyzed by a student t test, *** = p<0.001.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Western Blot

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: A) HEK293T cells were transfected with GFP-eVP40 (green) plus either vector, or BAG3-mCherry (red), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with arrows highlighting the typical localization pattern of GFP-eVP40 at the plasma membrane and in PM projections, while arrowheads highlight the altered diffuse cytoplasmic localization pattern of eVP40 observed in BAG3 expressing cells. Cell nuclei were stained with NucBlue. Scale bars = 10μm. B) HeLa cells were transfected with GFP-eVP40 (green) plus mCherry-LC3 (red) and vector alone (top row), or BAG3 (bottom two rows), and cells were imaged at 24 hours post transfection using a Leica SP5 FLIM inverted confocal microscope. Representative images are shown with white arrows highlighting the colocalization of GFP-eVP40 and mCherry-LC3 in aggresomes. Cell nuclei were stained with NucBlue. Scale bars = 10μm.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Plasmid Preparation, Microscopy, Expressing, Staining

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were mock-transfected or transfected with eVP40 (A) or mVP40 (C) plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 (B) or mVP40 (D) from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. E) HEK293T cells were transfected with eVP40 plus vector, BAG3-WT, or BAG3-ΔN as indicated. Cells were fixed at 24 hrs post-transfection, and then incubated with rabbit anti-eVP40 antiserum and mouse anti-myc antiserum (to detecting BAG3-WT and BAG3-ΔN). Cells were then stained with Alexa Fluor 488 goat anti-rabbit and 594 goat anti-mouse secondary antibodies. Microscopy was performed using a Leica SP5 FLIM inverted confocal microscope and XZY scanning. Representative images displaying eVP40 (green) and BAG3-WT (red) or BAG3-ΔN (red) localized at the PM are shown. Cell nuclei were stained with NucBlue. Scale bars = 10μm.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Isolation, Western Blot, Plasmid Preparation, Incubation, Staining, Microscopy

Journal: PLoS Pathogens

Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

doi: 10.1371/journal.ppat.1006132

Figure Lengend Snippet: HEK293T cells were first transfected with vector alone, BAG3-WT or BAG3-ΔN for 24 hours, and then infected with recombinant virus VSV-M40 (A) or VSV-M40-P2728A (C) at a MOI of 0.1 for 8 hours. Supernatants were harvested and virus titers were determined by standard plaque assay on BHK-21 cells. Each bar represents the average of three independent experiments performed in duplicate. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p<0.001. The indicated proteins from VSV-M40 (B) or VSV-M40(P2728A) (D) infected cell extracts were detected by Western blotting.

Article Snippet: The rabbit eVP40 antiserum (PROSCI), mouse anti-flag monoclonal antibody (SIGMA), mouse anti-GST monoclonal antibody (SIGMA) were used to detect eVP40-WT, eVP40-ΔPT/PY, mVP40, GST, or GST-BAG3 WW proteins in input and pulldown samples by Western blotting.

Techniques: Transfection, Plasmid Preparation, Infection, Recombinant, Plaque Assay, Western Blot