Journal: Neoplasia (New York, N.Y.)

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

doi: 10.1016/j.neo.2018.01.001

Figure Lengend Snippet: Overexpression of BAG3, HSP70 and other anti-apoptotic proteins and down-regulation of pro-apoptotic proteins in chemoresistant breast cancer cells. (A) BT-549 and MDA-MB-468 parental and chemoresistant cell lines were treated with 3 μM of apoptotic cell death inducer staurosporine (STS) and DMSO (0.1%) as control for 6 hours followed by % of total cell death and % of Annexin-V positive & PI negative cells were estimated by flow cytometry. Data are representative of three independent experiments performed in triplicate. Columns represent means ± SEM. Statistical significance; * p <0.05, ** p <0.01, *** p <0.001 and ns not significant compared to controls (DMSO). (B) Western blot analysis shows anti-apoptotic proteins (Bcl-2, Bcl-xL, and Mcl-1) are over expressed and pro-apoptotic proteins (Bax and Bak) are down regulated in chemoresistant cell lines compared to the parental counterparts. (C) BAG3 and (D) HSP70 are highly expressed in various chemoresistant cell lines. GAPDH was used as loading control. Densitometric analysis of relative BAG3 protein expression was performed in BT-549 and MDA-MB-468 parental and chemoresistant cell lines. Columns represent means ± SEM. Statistical significance; ** p<0.01, *** p<0.001 compared to parental control cells.

Article Snippet: Following primers were used for qPCR: TaqMan® Gene Expression Assay primer for BAG3 (Hs00188713_m1), CDH1 (Hs01023895_m1), CDH2 (Hs00169953_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), TWIST1 (Hs01675818_S1), TWIST2 (Hs02379973_s1).

Techniques: Over Expression, Control, Flow Cytometry, Western Blot, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

doi: 10.1016/j.neo.2018.01.001

Figure Lengend Snippet: Depletion of BAG3 sensitizes the chemoresistant breast cancer cells by attenuating protective autophagy (A) Stable lentiviral knockdowns of BAG3 were established in both the BT-549-Par and BT-549 r DOX 20 cells and (B) also in the MDA-MB-468Par and MDA-MB-468 r 5-FU 2000 cells. Stable lentiviral transduction of empty vector was used as control (Ctrl). Knockdowns were confirmed by western blot. Knockdown of BAG3 reduced LC3-II protein expression in BT-549 r DOX 20 and MDA-MB-468 r 5-FU 2000 cells. GAPDH served as loading control in western blot. (C) Relative BAG3 mRNA expression was also determined by quantitative PCR (qPCR) after stable lentiviral knockdowns of BAG3 in BT-549Par and BT-549 r DOX 20 cell lines and (D) similarly in MDA-MB-468 cells respectively. qPCR data represent means of three independent experiments± SEM (n = 3). Significant BAG3 mRNA expression compared to parental sh Ctrls are marked by asterisks : * p<0.05, ** p<0.01 and ns not significant. Significant differences between BAG3 KD and respective sh Ctrls are denoted by hashtags : # p<0.05, ## p <0.01 and ns not significant. (E) Knockdown of BAG3 reduced the protein expression of HSP70, Mcl-1 and other anti-apoptotic proteins whereas augments the expression of pro-apoptotic proteins like Bak and Bax in BT-549 r DOX 20 and (F) MDA-MB-468 r 5-FU 2000 cells respectively. GAPDH served as loading control. (G) BT-549 r DOX 20 /BAG3 KD and (H) MDA-MB-468 r 5-FU 2000 /BAG3 KD cells exhibited significantly higher levels of total cell death compared to their parental counterparts after 72 hours of DOX and 5-FU treatment in a dose dependent manner respectively. 0.1% DMSO for 72 h was used as control. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Data represent means of three independent experiments ± SEM (n = 3). * p <0.05 and ns not significant of BAG3 KD compared to respective sh Ctrls.

Article Snippet: Following primers were used for qPCR: TaqMan® Gene Expression Assay primer for BAG3 (Hs00188713_m1), CDH1 (Hs01023895_m1), CDH2 (Hs00169953_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), TWIST1 (Hs01675818_S1), TWIST2 (Hs02379973_s1).

Techniques: Transduction, Plasmid Preparation, Control, Western Blot, Knockdown, Expressing, Real-time Polymerase Chain Reaction, Double Staining, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

doi: 10.1016/j.neo.2018.01.001

Figure Lengend Snippet: HSP70/BAG3 interaction inhibitor YM-1 and HSF-1 inhibitor KRIBB11 sensitize the chemoresistant breast cancer cells (A) Dissociation of the HSP70/BAG3 complex by YM-1 (5 μM) decreased Mcl-1 expression in BT-549 r DOX 20 cells after 4 h, 18 h, 24 h, 48 h treatment in western blot analysis. 0.1% DMSO for 48 h was used as Control (Ctrl) for the solvent, whereas Sorafenib (Sora) 5 μM for 48 h was used as positive control. GAPDH was used as loading control in western blot. (B) % of cell viability was analyzed by MTT assay in BT-549Par and BT-549 r DOX 20 cells after 2 h pre-treatment of 5 μM of YM-1 for 48 h followed by DOX for 72 h. DMSO 0.1 % for 48 h was used as control (Ctrl) for the solvent. (C) Breast cancer cell lines BT-549Par and BT-549 r DOX 20 were treated with 10 ng/ml and 80 ng/ml of DOX for 72 h with or without YM-1 (5 μM, 48 h). 0.1 % DMSO for 48 h was used as control (Ctrl) for the solvent. Then cell death was analyzed by Annexin-V positive staining in flow cytometry. Columns represent means of three independent experiments ± SEM (n = 3). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % DMSO); # p<0.05, ## p<0.01 and ns not significant with combined treatment of YM-1 and DOX compared to DOX treatment alone. (D) Combined treatment of KRIBB11 (20 μM, 48 h) and DOX significantly augment the total cell death in BT-549 r DOX 20 /sh Ctrl cells compared to DOX treatment alone. 0.1 % DMSO for 48 h was used as control (Ctrl) for the solvent. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments ± SEM (n = 3). Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % DMSO); # p<0.05, ## p<0.01 and ns not significant with combined treatment of KRIBB11 and DOX compared to DOX treatment alone. (E) KRIBB11 treatment decreased BAG3, HSP70, Mcl-1 and LC3 II in a dose dependent manner in BT-549 r DOX 20 and (F) MDA-MB-468 r 5-FU 2000 cells respectively. Cells were treated with 5 μM, 10 μM and 20 μM of KRIBB11 for 48 h. DMSO (0.1 %, 48 h) was used as control (Ctrl). GAPDH served as loading control for western blot.

Article Snippet: Following primers were used for qPCR: TaqMan® Gene Expression Assay primer for BAG3 (Hs00188713_m1), CDH1 (Hs01023895_m1), CDH2 (Hs00169953_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), TWIST1 (Hs01675818_S1), TWIST2 (Hs02379973_s1).

Techniques: Expressing, Western Blot, Control, Solvent, Positive Control, MTT Assay, Staining, Flow Cytometry, Double Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

doi: 10.1016/j.neo.2018.01.001

Figure Lengend Snippet: BAG3 depletion alters cell matrix adhesion in chemoresistant breast cancer cells (A) Knockdown of BAG3 altered cellular morphology and actin cytoskeletal distribution in BT-549 r DOX 20 and (B) MDA-MB-468 r 5-FU 2000 cells compared to their respective parental counterparts in confocal imaging. For staining of F-actin, Texas Red-X phalloidin was used whereas nuclei were stained with DAPI. Scale bar 10 μm. (C) FAK phosphorylation was reduced in BT-549 r DOX 20 /BAG3 KD and (D) MDA-MB-468 r 5-FU 2000 /BAG3 KD cells respectively. GAPDH served as loading control in the western blot. Densitometric analysis of relative pFAK protein expression was performed in control and BAG3 KD of both BT-549 and MDA-MB-468 parental and chemoresistant cell lines. Columns represent means ± SEM. Statistical significance; * p<0.05, ** p<0.01, *** p<0.001 compared to controls (E) BT-549 r DOX 20 /BAG3 KD cells cultured in suspension exhibited more sensitivity to DOX treatment in a dose dependent manner. Cell cultured dishes were coated with pHEMA to prevent cell adhesion. Water (0.1%, 72 h) was used as control (Ctrl) for the solvent. Cell death was determined by Annexin V/PI double staining followed by flow cytometry. Columns represent means of three independent experiments performed in triplicate ± SEM. Statistical significance: * p<0.05, ** p<0.01, *** p<0.001 and ns not significant compared to ctrls (0.1 % water); # p<0.05 and ns not significant with combined treatment of DOX and pHEMA compared to DOX treatment alone.

Article Snippet: Following primers were used for qPCR: TaqMan® Gene Expression Assay primer for BAG3 (Hs00188713_m1), CDH1 (Hs01023895_m1), CDH2 (Hs00169953_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), TWIST1 (Hs01675818_S1), TWIST2 (Hs02379973_s1).

Techniques: Knockdown, Imaging, Staining, Phospho-proteomics, Control, Western Blot, Expressing, Cell Culture, Suspension, Solvent, Double Staining, Flow Cytometry

Journal: Neoplasia (New York, N.Y.)

Article Title: BAG3 Overexpression and Cytoprotective Autophagy Mediate Apoptosis Resistance in Chemoresistant Breast Cancer Cells

doi: 10.1016/j.neo.2018.01.001

Figure Lengend Snippet: Knockdown of BAG3 reverses the EMT phenomena and simultaneously suppresses invasion in chemoresistant breast cancer cells (A) Knockdown of BAG3 reduced the relative CDH2 , SNAI1 mRNA expression, simultaneously increased CDH1 mRNA expression in BT-549 r DOX 20 /BAG3 KD and (B) MDA-MB-468 r 5-FU 2000 /BAG3 KD cells in qPCR respectively. qPCR data represent means of three independent experiments ± SEM (n = 3). Significant mRNA expression compared to parental sh Ctrls are marked by asterisks : * p<0.05, ** p<0.01 and ns not significant. Significant differences between BAG3 KD and respective sh Ctrls are denoted by hashtags : # p<0.05, ## p <0.01 and ns not significant. (C) Expression of E-cadherin protein was increased whereas N-cadherin expression was decreased in MDA-MB-468 r 5-FU 2000 /BAG3 KD cells in western blot. GAPDH served as loading control. (D) Number of invaded cells was decreased in BT-549 r DOX 20 /BAG3 KD and (E) MDA-MB-468 r 5-FU 2000 /BAG3 KD cells. Invasion assay was performed for 20 h followed by stained with 1% crystal violet, bright field image was taken and invaded cells were counted by using ImageJ software. Columns represent means of three independent experiments ± SEM (n = 3). Statistical significance of invasion: ** p<0.01, *** p<0.001 and ns not significant with BAG3 KD compared to sh Ctrls.

Article Snippet: Following primers were used for qPCR: TaqMan® Gene Expression Assay primer for BAG3 (Hs00188713_m1), CDH1 (Hs01023895_m1), CDH2 (Hs00169953_m1), SNAI1 (Hs00195591_m1), SNAI2 (Hs00161904_m1), TWIST1 (Hs01675818_S1), TWIST2 (Hs02379973_s1).

Techniques: Knockdown, Expressing, Western Blot, Control, Invasion Assay, Staining, Software

Journal: Journal of cell science

Article Title: 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes.

doi: 10.1242/jcs.126102

Figure Lengend Snippet: Fig. 7. 14-3-3 interacts with chaperone proteins Hsp70 and BAG3. Western blot analyses of 14-3-3 immunoprecipitates and cell lysates from tsA201 cells expressing various combinations of proteins as indicated. (A) BAG3 and 14-3-3cHA co-immunoprecipitate from co-transfected tsA201 cells (lane 2), and the extent of BAG3-14-3-3 interaction is not altered by exogenously expressed Hsp70 (lane 1). (B) Hsp70 co-immunoprecipitates with 14-3-3cHA in co-transfected cells (lane 2), but its binding to 14-3-3 is increased when co-transfected with BAG3 (lane 1). (C) Residues S173 and S136 in BAG3 are necessary for 14-3-3 binding. Compared with WT (lane 5), the S136A BAG3 has reduced binding to 14-3-3 (lane 1), whereas the S173A mutation abolishes the interaction completely (lane 3). (D) Interaction between 14-3-3 and BAG3 is regulated by phosphorylation. (Left panels) co-immunoprecipitation of 14-3-3 and BAG3 is reduced by alkaline phosphatase (AP) treatment of cell lysates. (Right panels) In vitro phosphorylation of recombinant BAG3 with crude tsA201 cell extract significantly enhances its binding to 14-3-3c, as assessed by co-immunoprecipitation and western blotting. For controls, either ATP (lane 2) or recombinant 14-3-3c (lane 3) was omitted.

Article Snippet: The following antibodies were used in this study: polyclonal anti-a-Syn antibody (#2642, Cell Signaling Technology), monoclonal anti-vimentin antibody (#6630, Sigma-Aldrich), homemade monoclonal anti-HA antibody, monoclonal anti-flag M2 antibody (Sigma-Aldrich), monoclonal anti-dynein (intermediate chain) antibody (D5167, Sigma-Aldrich), monoclonal anti-GAPDH antibody (MCA-1D4, Stemcell Technologies), monoclonal anti-GFP antibody (NeuroMab), polyclonal anti-GFP antibody (sc-8334, Santa Cruz), polyclonal anti-BAG3 antibody (Proteintech), polyclonal anti-HSC70/HSP70 antibody (pAb, Enzo Life Sciences), polyclonal anti-14-3-3c antibody (C-16, Santa Cruz), homemade monoclonal anti-1D4 antibody, polyclonal anti-Giantin antibody (Abcam), polyclonal IRDye 800CW Conjugated Goat anti-mouse IgG (H+L, LI-COR) and polyclonal IRDye 800CW Conjugated Goat anti-rabbit IgG (H+L, LI-COR).

Techniques: Western Blot, Expressing, Transfection, Binding Assay, Mutagenesis, Phospho-proteomics, Immunoprecipitation, In Vitro, Recombinant

Journal: Journal of cell science

Article Title: 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes.

doi: 10.1242/jcs.126102

Figure Lengend Snippet: Fig. 8. 14-3-3 is crucial for associations of BAG3 and cargos with dynein. Western blot analyses of GST pull-down assays and cell lysates from tsA201 cells expressing various combinations of proteins as indicated. (A) The BAG3–DIC association is enhanced by exogenously expressed 14-3-3cHA (lane 2), but eliminated by co-transfection of pSCM138 (lane 3). (B) Compared with WT BAG3, the S136A BAG3 mutant has a reduced binding to DIC, and the S173A BAG3 mutant does not co-precipitate with GST-mDIC. (C) Binding of recombinant GST-mDIC and BAG3 is enhanced by in vitro phosphorylation of BAG3 with tsA201 cell extract (lane 1). This effect is not observed when 14-3-3 (lane 2) or ATP (lanes 3, 4) is absent. (D) The association of SODG85R-GFP with GST-mDIC is enhanced by exogenously expressed 14-3-3cHA (lane 3), and virtually abolished when pSCM138 (lane 1) is co- transfected.

Article Snippet: The following antibodies were used in this study: polyclonal anti-a-Syn antibody (#2642, Cell Signaling Technology), monoclonal anti-vimentin antibody (#6630, Sigma-Aldrich), homemade monoclonal anti-HA antibody, monoclonal anti-flag M2 antibody (Sigma-Aldrich), monoclonal anti-dynein (intermediate chain) antibody (D5167, Sigma-Aldrich), monoclonal anti-GAPDH antibody (MCA-1D4, Stemcell Technologies), monoclonal anti-GFP antibody (NeuroMab), polyclonal anti-GFP antibody (sc-8334, Santa Cruz), polyclonal anti-BAG3 antibody (Proteintech), polyclonal anti-HSC70/HSP70 antibody (pAb, Enzo Life Sciences), polyclonal anti-14-3-3c antibody (C-16, Santa Cruz), homemade monoclonal anti-1D4 antibody, polyclonal anti-Giantin antibody (Abcam), polyclonal IRDye 800CW Conjugated Goat anti-mouse IgG (H+L, LI-COR) and polyclonal IRDye 800CW Conjugated Goat anti-rabbit IgG (H+L, LI-COR).

Techniques: Western Blot, Expressing, Cotransfection, Mutagenesis, Binding Assay, Recombinant, In Vitro, Phospho-proteomics, Transfection

Journal: Journal of cell science

Article Title: 14-3-3 protein targets misfolded chaperone-associated proteins to aggresomes.

doi: 10.1242/jcs.126102

Figure Lengend Snippet: Fig. 9. The interaction between 14-3-3 and BAG3 is required for aggresome formation. (A) Aggresome formation promoted by 14-3-3 is impaired in cells treated with siRNA targeting Bag3. The bar graph shows GFP-CFTRDF508 aggresome formation in control cells (white bars) or BAG3-knockdown cells (gray bars) co-transfected with 14-3-3cHA, empty vector or pSCM138. Representative confocal images of aggresomes in control cells and cells treated with BAG3 siRNA are shown on the right. Scale bar: 10 mm. (B) Percentage of cells containing aggresomes in BAG3- knockdown cells that had BAG3 reintroduced through transfection of BAG3-siRNA-resistant wild-type BAG3 (WT BAG3). (C) Percentage of cells that contain aggresomes in cells that had been transfected with BAG3-siRNA (to knock down endogenous Bag3) and a 14-3-3-binding- deficient Bag3 mutant (S136A/S173A BAG3) that is resistant to BAG3 siRNA (mutant BAG3) (*P,0.05, n55). Insets in B and C show the levels of exogenously expressed BAG3-siRNA-resistant wild-type BAG3 (WT BAG3) and BAG3-siRNA-resistant 14-3-3-binding-deficient BAG3 (mutant BAG3), respectively, in cells treated with BAG3 siRNA.

Article Snippet: The following antibodies were used in this study: polyclonal anti-a-Syn antibody (#2642, Cell Signaling Technology), monoclonal anti-vimentin antibody (#6630, Sigma-Aldrich), homemade monoclonal anti-HA antibody, monoclonal anti-flag M2 antibody (Sigma-Aldrich), monoclonal anti-dynein (intermediate chain) antibody (D5167, Sigma-Aldrich), monoclonal anti-GAPDH antibody (MCA-1D4, Stemcell Technologies), monoclonal anti-GFP antibody (NeuroMab), polyclonal anti-GFP antibody (sc-8334, Santa Cruz), polyclonal anti-BAG3 antibody (Proteintech), polyclonal anti-HSC70/HSP70 antibody (pAb, Enzo Life Sciences), polyclonal anti-14-3-3c antibody (C-16, Santa Cruz), homemade monoclonal anti-1D4 antibody, polyclonal anti-Giantin antibody (Abcam), polyclonal IRDye 800CW Conjugated Goat anti-mouse IgG (H+L, LI-COR) and polyclonal IRDye 800CW Conjugated Goat anti-rabbit IgG (H+L, LI-COR).

Techniques: Control, Knockdown, Transfection, Plasmid Preparation, Binding Assay, Mutagenesis

Journal: Clinical and Translational Science

Article Title: Identification of BAG2 and Cathepsin D as Plasma Biomarkers for Parkinson’s Disease

doi: 10.1111/cts.12920

Figure Lengend Snippet: Average protein levels of BAG2 and cathepsin D are decreased in the plasma of PD patients. ( a ) Preparation of the albumin/IgG‐depleted plasma samples from control and PD patients (20 subjects each) was verified by SDS‐PAGE. The plasma samples from the 20 control and patients with PD were each pooled for the quantitative analysis of the average levels of autophagy‐related proteins. The pooled samples were immunoblotted with antibodies specific to BAG2 ( b ), BAG3 ( c ), cathepsin B ( d ), cathepsin D ( e ), LAMP1 ( f ), LAMP2 ( g ), and WDFY3 ( h ). Each analysis was repeated as necessary. Arrows and asterisks indicate specific and cross‐reacting bands, respectively. ( i ) Intensities of specific bands of b–h were quantitatively analyzed, and the level of changes in the PD group was compared to that in the control group. Each blue dot indicates the result from each blot, and the grey bars represent mean values. ( j ) The fold change in BAG2 levels of the PD group. Values represent mean ± SEM of three independent experiments. ( k ) The fold change in cathepsin D levels of the PD group. Values represent mean ± SEM of two independent experiments. Molecular weight standards (in kDa) are shown to the left. ** P < 0.01 (Student t ‐test). BAG, BCL2‐associated athanogene; CBB, Coomassie Brilliant Blue; PD, Parkinson’s disease; SDS‐PAGE, sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.

Article Snippet: Primary antibodies specific to BCL2‐associated athanogene (BAG2; Bethyl Cat #A304‐751A), BAG3 (Bethyl Cat #A302‐807A), cathepsin B (Abcam Cat #ab58802), cathepsin D (Calbiochem Cat #IM‐03), LAMP1 (Abcam Cat #ab25630), LAMP2 (Proteintech Cat #66301‐1‐Ig), WDFY3 (Bethyl Cat #A301‐869A), and α‐Tubulin (Abcam Cat #ab18251) were purchased.

Techniques: Clinical Proteomics, Control, SDS Page, Molecular Weight, Polyacrylamide Gel Electrophoresis

Journal: Circulation

Article Title: BAG3 (Bcl-2–Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy

doi: 10.1161/circulationaha.116.024873

Figure Lengend Snippet: Figure 2. Strain-specific coding variants of BAG3 differentially promote limb muscle

Article Snippet: The following commercial antibodies were used: FLAG, 123 LC3b, ATG7, Beclin, HspB8, SQSTM1/p62 (Cell Signaling), BAG3 (Polyclonal, Imgenex), 124 GAPDH (Novus Biologicals), tubulin (Santa Cruz), CD31 (Abd Serotec MCA-1364), SMA 125 (DAKO, 1A4).

Techniques:

Journal: Cell reports

Article Title: Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy

doi: 10.1016/j.celrep.2023.113529

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: rabbit anti-Bag3 , Novus , Cat # NBP2–27398SS.

Techniques: Recombinant, Injection, Western Blot, Cell Culture, Stripping Membranes, Labeling, Plasmid Preparation, shRNA, Software, Fluorescence, Microscopy

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: pVI of HAdV-B7 interacted with the host protein BAG3. A HEK 293T cells were transfected with plasmids encoding BAG3-HA (2 ​μg) and pVI-Flag (2 ​μg) or vector-Flag (2 ​μg) for 24 ​h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. B HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 ​μg) and BAG3-HA (2 ​μg) or vector-HA (2 ​μg) for 24 ​h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. C A549 ​cells were transfected with plasmids encoding BAG3-HA (2 ​μg) and pVI-Flag (2 ​μg) or vector-Flag (2 ​μg) for 24 ​h, followed by Co-IP with anti-Flag binding beads and immunoblot analysis with the indicated antibodies. D A549 ​cells were transfected with plasmids encoding pVI-Flag (2 ​μg) and BAG3-HA (2 ​μg) or vector-HA (2 ​μg) for 24 ​h, followed by Co-IP with anti-HA binding beads and immunoblot analysis with the indicated antibodies. E HEK 293T cells were transfected with vector-Flag (2 ​μg) or pVI-Flag (2 ​μg). Cell lysates were evaluated by Western blotting using specific antibodies against BAG3 and LC3. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. ∗, P ​< ​0.05. F HEK 293T cells were transfected with plasmids encoding pVI-Flag (2 ​μg) and BAG3-HA (2 ​μg), followed by labeling Flag, HA and LAMP1 with a specific primary antibody. The cell nucleus were stained with DAPI. Fluorescence signals were observed using confocal immunofluorescence microscopy. G Quantitative analysis of colocalized immunofluorescence intensity was using ImageJ, and approximately 25–30 ​cells in total for each condition were used for quantification. ∗, P ​< ​0.05. Scale bars: 5 ​μm.

Article Snippet: Rabbit anti-SQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Expressing, Labeling, Staining, Fluorescence, Immunofluorescence, Microscopy

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: BAG3 interacted with pVI in a WW domain-dependent manner. A Schematic diagram of BAG3-WT and the truncation mutants BAG3-ΔN and BAG3-ΔC, highlighting the locations of the functional domains, including the N-terminal WW domain (blue) and the C-terminal BAG domain (red). All three proteins contained HA tags. B HEK 293T cells transfected with the indicated plasmid (2 ​μg) and BAG3-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or truncation mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI was detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody.

Article Snippet: Rabbit anti-SQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

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

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: BAG3 interacted with the PPSY structural domain of pVI through its WW structural domain. A Schematic diagram of pVI-WT, pVI truncation mutants, and PPSY mutants (PAGG). All five proteins contained Flag tags. B HEK 293T cells transfected with the indicated plasmid (2 ​μg) and pVI-WT or truncation mutant proteins were detected by Western blotting. C Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-ΔC was detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. D Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody. E Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-Flag binding beads. BAG3-WT or mutant proteins were detected in the precipitates by Western blotting using an anti-HA-specific primary antibody. F Extracts from HEK 293T cells transfected with the indicated plasmid combinations were first immunoprecipitated with anti-HA binding beads. pVI 1–170 was detected in the precipitates by Western blotting using an anti-Flag-specific primary antibody.

Article Snippet: Rabbit anti-SQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Plasmid Preparation, Mutagenesis, Western Blot, Immunoprecipitation, Binding Assay

Journal: Virologica Sinica

Article Title: Autophagy induced by human adenovirus B7 structural protein VI inhibits viral replication

doi: 10.1016/j.virs.2023.08.002

Figure Lengend Snippet: BAG3-induced autophagy inhibited viral replication. A549 cells ( A ) and 16HBE cells ( B ) were transfected with si BAG3 or siNC. Cell lysates were subjected to Western blotting analysis 24 ​h after transfection. A549 cells ( C ) and 16HBE cells ( D ) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 ​h. Cell lysates were subjected to Western blotting analysis at 24 ​h after transfection to detect the expression levels of BAG3, p62, LC3, DBP by using specific antibodies. Representative immunoblots were showed and densitometric analysis of the relative protein expression were from three independent experiments. Statistical significance was analyzed by Student's t -test, ∗, P ​< ​0.05; ∗∗, P ​< ​0.01. A549 cells ( E ) and 16HBE cells ( F ) were transfected with si BAG3 or siNC. Cell lysates were subjected to qPCR analysis using E1A -specific primers. A549 cells ( G ) and 16HBE cells ( H ) were transfected with plasmids encoding BAG3-HA or vector-HA for 24 ​h. Cell lysates were subjected to qPCR analysis using E1A -specific primers. Quantification of the relative mRNA levels of E1A from three independent experiments. Statistical significance was analyzed by Student's t -test, ∗∗, P ​< ​0.01; ∗∗∗, P ​< ​0.001; ∗∗∗∗, P ​< ​0.0001.

Article Snippet: Rabbit anti-SQSTM1/p62 antibody (5114S), rabbit anti-BAG3 antibody (23842), mouse anti-LAMP1 antibody (15665), rabbit anti-DYKDDDDK/FLAG tag antibody (14793), and rabbit anti-Atg5 antibody (12994) were purchased from Cell Signaling Technology (Massachusetts, USA).

Techniques: Transfection, Western Blot, Plasmid Preparation, 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 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