Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Plasmids used in the assays

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Expressing

Journal: Intervirology

Article Title: The Regulation of Prototype Foamy Virus 5′Long Terminal Repeats and Internal Promoter by Endogenous Transcription Factors

doi: 10.1159/000517539

Figure Lengend Snippet: Promoter activities of PFV 5′LTR and IP regulated by AP-1 (a) and BAG3 (b). The plasmids pcDNA-jun and pcDNA-fos were used to express the 2 subunits of the heterodimer AP-1. The plasmid pcDNA-BAG3 was used to express the BAG3 protein. At the bottom of each column, the amount (nanogram) of the plasmids co-transfected in each group was labeled. PFV, prototype foamy virus; LTR, long terminal repeat; IP, internal promoter; TRE, Tas responsive elements; AP-1, activator promoter-1; PMA, phorbol-12-myristate-13-acetate; BAG3, BCL2-associated athanogene 3. Asterisks indicate significant difference.

Article Snippet: Then the proteins were transferred to PVDF membrane and incubated with anti-BAG3 (Bioss, China) and anti-fos (Bioss) polyclonal antibodies, respectively.

Techniques: Plasmid Preparation, Transfection, Labeling

Journal: Nature Communications

Article Title: BAG3 promotes pancreatic ductal adenocarcinoma growth by activating stromal macrophages

doi: 10.1038/ncomms9695

Figure Lengend Snippet: Peritoneal metastasis inhibition by anti-BAG3 mAb.

Article Snippet: Anti-BAG3 rabbit pAb (polyclonal Antibody) and murine mAbs (monoclonal Antibodies) and their F(ab') 2 fragments were obtained from BIOUNIVERSA s.r.l., SA, Italy.

Techniques: Inhibition, Control

Journal: Nature cell biology

Article Title: TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin binding proteins

doi: 10.1038/s41556-025-01789-5

Figure Lengend Snippet: ( a ) Expression of BAG3 in the adult mouse spinal cord. The image is ISH data from the Allen mouse spinal cord atlas 46 https://mousespinal.brain-map.org/imageseries/show.html?id=100020020 and depicts a cervical slice from a P56 mouse. ( b ) Representative live images of cells expressing BAG3 mRuby2 and TDP-43 ΔNTD-Clover or TDP-43 ΔNTD-Clover under MG132 treatment. ( c ) Quantification of cells with skein-like inclusions at different time points. N=109, 185, 111, 118 cells at 2 hr and 105, 127, 105, 111 cells at 3 hr (TDP-43 ΔNTD-Clover ) per replicate and N=102, 122, 134, 190 cells at 2 hr and 105, 131, 130, 128 cells at 3 hr (BAG3 mRuby2 and TDP-43 ΔNTD-Clover ) per replicate. ( d ) Representative images of BAG3 and TDP-43 ΔNTD-Clover in cells under no stress, 100 μM NaAsO 2 or 10 μM MG132. Cells were permeabilized with 50 μg/mL digitonin for 5 mins before fixation. ( e ) Representative images of HSPB1, HSPB8 and TDP-43 ΔNTD-Clover in cells under no stress, 100 μM NaAsO 2 or 10 μM MG132. Cells were permeabilized with 50 μg/mL digitonin for 5 mins before fixation. ( f ) Representative live images of TDP-43 ΔNTD-Clover skein-like or granule-like inclusions following 100 μM NaAsO 2 treatment in the cells transfected with control siRNA, siBAG3, siHSPB8 or siHSPB1. ( g - h ) Quantification of cells with granule-like or skein-like inclusions under the conditions described in ( f ). N: four biological replicates. Cell number quantified in each replicate experiment: 183, 175, 192 (control siRNA); 159, 192, 160 (siBAG3); 225, 201, 176 (siHSPB8); 123, 143, 154 (siHSPB1). Bar: SD.

Article Snippet: Then membrane was blocked with Intercept Blocking Buffer (LICOR) and incubated with primary antibodies: BAG3 rabbit polyclonal antibody, Novus Biologicals, NBP2–27398, Lot#102119, at 1:1000 dilution, HSPB8 rabbit polyclonal antibody, Stressgen, NBP2–87836, at 1:1000, filamin A mouse monoclonal antibody, Sigma, MAB1680-C, at 1:1000, Actinin rabbit polyclonal antibody, Proteintech, #11313–2-AP) at 1:1000, HSPB1 monoclonal antibody, StressMarq, 5D12-A12 at 1:1000 dilution, HSP70 monoclonal antibody, Enzo ADI-SPA-810 at 1:1000 dilution, GAPDH polyclonal antibody, Cell Signaling 14C10, 1:2000 dilution and mouse monoclonal, Abcam, ab8245, TDP-43 C-terminal antibody, Proteintech, 12892–1-AP at 1:1000 dilution, actin rabbit antibody, Abcam, ab8227, at 1:1000 dilution, overnight at 4 °C.

Techniques: Expressing, Transfection, Control

Journal: Nature cell biology

Article Title: TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin binding proteins

doi: 10.1038/s41556-025-01789-5

Figure Lengend Snippet: ( a ) Schematic diagram illustrating the biochemical analysis of soluble and insoluble fractions of cells expressing TDP-43 ΔNTD-Clover or Clover. ( b ) Analysis of the levels of TDP-43 ΔNTD-Clover , endogenous TDP-43, FLNA, β-Actin, Actinin, BAG3, HSPA1A, GAPDH in the soluble fractions of cells under no stress, 100 μM NaAsO 2 or 10 μM MG132 treatment. Data are from three biological replicates. ( c ) Analysis of the levels of TDP-43 ΔNTD-Clover , endogenous TDP-43, FLNA, β-Actin, Actinin, BAG3, HSPA1A, GAPDH in the insoluble fractions of cells under no stress, 100 μM NaAsO 2 or 10 μM MG132 treatment. Data are from three biological replicates. ( d ) Quantification of the levels of TDP-43 ΔNTD-Clover , endogenous TDP-43 and FLNA in both soluble and insoluble fractions. n=3 independent replicates. Error bar: SD. ( e ) Specific interaction of TDP-43 with FLNA and actin under NaAsO 2 or MG132 stress conditions detected by immunoprecipitation.

Article Snippet: Then membrane was blocked with Intercept Blocking Buffer (LICOR) and incubated with primary antibodies: BAG3 rabbit polyclonal antibody, Novus Biologicals, NBP2–27398, Lot#102119, at 1:1000 dilution, HSPB8 rabbit polyclonal antibody, Stressgen, NBP2–87836, at 1:1000, filamin A mouse monoclonal antibody, Sigma, MAB1680-C, at 1:1000, Actinin rabbit polyclonal antibody, Proteintech, #11313–2-AP) at 1:1000, HSPB1 monoclonal antibody, StressMarq, 5D12-A12 at 1:1000 dilution, HSP70 monoclonal antibody, Enzo ADI-SPA-810 at 1:1000 dilution, GAPDH polyclonal antibody, Cell Signaling 14C10, 1:2000 dilution and mouse monoclonal, Abcam, ab8245, TDP-43 C-terminal antibody, Proteintech, 12892–1-AP at 1:1000 dilution, actin rabbit antibody, Abcam, ab8227, at 1:1000 dilution, overnight at 4 °C.

Techniques: Binding Assay, Expressing, Immunoprecipitation

Journal: Nature cell biology

Article Title: TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin binding proteins

doi: 10.1038/s41556-025-01789-5

Figure Lengend Snippet: ( a ) Representative images of motor neurons from ALS patients displaying different types of TDP-43 pathological structures. A dot-plot quantifies the percentage of motor neurons exhibiting different types of TDP-43 pathological structures. Number of motor neurons quantified are 400, 164, 114, 105, 90, 144, 70, 70, 80 from nine patients. Error bar: SD. ( b ) Schematic representation of the interaction domains of HSP70, BAG3 and HSPB8, HSPB1. ( c - e ) Expression levels of BAG1, BAG2, BAG3, BAG4, BAG5, BAG6 and HSPH1 in different cell types within the adult human spinal cord. Data are adapted from scRNA-seq datasets published by Yadav, et al., in Nature Neuroscience 44 ( c , d ) and by Gautier, et al., in Neuron 45 ( e ). Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. ( f ) Expression levels of BAG1, BAG2, BAG4, BAG5 and HSPH1 in different cell types within the adult mouse spinal cord. Images are ISH data from the Allen mouse spinal cord atlas ( https://mousespinal.brainmap.org/ , https://mousespinal.brainmap.org/imageseries/show.html?id=100024593 , https://mousespinal.brain-map.org/imageseries/show.html?id=100038126 , https://mousespinal.brain-map.org/imageseries/show.html?id=100037229 , https://mousespinal.brain-map.org/imageseries/show.html?id=100048120 , https://mousespinal.brain-map.org/imageseries/show.html?id=100021840 ).

Article Snippet: Then membrane was blocked with Intercept Blocking Buffer (LICOR) and incubated with primary antibodies: BAG3 rabbit polyclonal antibody, Novus Biologicals, NBP2–27398, Lot#102119, at 1:1000 dilution, HSPB8 rabbit polyclonal antibody, Stressgen, NBP2–87836, at 1:1000, filamin A mouse monoclonal antibody, Sigma, MAB1680-C, at 1:1000, Actinin rabbit polyclonal antibody, Proteintech, #11313–2-AP) at 1:1000, HSPB1 monoclonal antibody, StressMarq, 5D12-A12 at 1:1000 dilution, HSP70 monoclonal antibody, Enzo ADI-SPA-810 at 1:1000 dilution, GAPDH polyclonal antibody, Cell Signaling 14C10, 1:2000 dilution and mouse monoclonal, Abcam, ab8245, TDP-43 C-terminal antibody, Proteintech, 12892–1-AP at 1:1000 dilution, actin rabbit antibody, Abcam, ab8227, at 1:1000 dilution, overnight at 4 °C.

Techniques: Expressing, Software

Journal: Nature cell biology

Article Title: TDP-43 skein-like inclusions are formed by BAG3- and HSP70-guided co-aggregation with actin binding proteins

doi: 10.1038/s41556-025-01789-5

Figure Lengend Snippet: ( a ) Representative images showing the localization of endogenous BAG3 in cells stained with BAG3 antibody from more than three biological replicates. ( b ) Western blot analysis of BAG3 in cells without the mRuby2-tagged BAG3 transgene or in cells stably expressing BAG3 mRuby2 or BAG3 R480A-mRuby2 . ( c - d ) Western blot demonstrating the reduction of BAG3 ( c ) and HSPB1 ( d ) following siRNA treatment. ( e ) Decrease of HSPB8 following siRNA treatment detected by immunostaining and quantification of mean fluorescent intensity in cells. Volcano plots show the mean fluorescent intensities of HSPB8 and HSPB1 in cells treated with siHSPB8 or control siRNA. Center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, outliers are represented by dots. ( f ) Schematic illustrating the assessment of BAG3 association with TDP-43 ΔNTD-Clover on skein-like structures. ( g - h ) Representative live images of cells co-expressing TDP-43 ΔNTD-Clover with BAG3 mRuby2 ( g ) or BAG3 R480A-mRuby2 ( h ) upon 100 μM NaAsO 2 treatment. ( i ) Measurement of HSP70 activity before, during, and after the removal of arsenite stress from more than three biological replicates. Error bar: SD. ( j ) The effect of expressing BAG3 mRuby2 and BAG3 R480A-mRuby2 on cell resistance to HSP70 inhibition. Data are from N=3 biological replicates. Number of quantified cells per replicate are 136, 168 and 202 (TDP-43 ΔNTD-Clover , no Dox); 140, 140, 156 (TDP-43 ΔNTD-Clover , Dox); 275, 286, 258 (TDP-43 ΔNTD-Clover and BAG3 mRuby2 , no Dox); 234, 178, 233 (TDP-43 ΔNTD-Clover and BAG3 mRuby2 , Dox); 198, 161, 210 (TDP-43 ΔNTD-Clover and BAG3 R480A-mRuby2 , no Dox); 175, 166, 164 (TDP-43 ΔNTD-Clover and BAG3 R480A-mRuby2 , Dox), respectively. Error bar: SD.

Article Snippet: Then membrane was blocked with Intercept Blocking Buffer (LICOR) and incubated with primary antibodies: BAG3 rabbit polyclonal antibody, Novus Biologicals, NBP2–27398, Lot#102119, at 1:1000 dilution, HSPB8 rabbit polyclonal antibody, Stressgen, NBP2–87836, at 1:1000, filamin A mouse monoclonal antibody, Sigma, MAB1680-C, at 1:1000, Actinin rabbit polyclonal antibody, Proteintech, #11313–2-AP) at 1:1000, HSPB1 monoclonal antibody, StressMarq, 5D12-A12 at 1:1000 dilution, HSP70 monoclonal antibody, Enzo ADI-SPA-810 at 1:1000 dilution, GAPDH polyclonal antibody, Cell Signaling 14C10, 1:2000 dilution and mouse monoclonal, Abcam, ab8245, TDP-43 C-terminal antibody, Proteintech, 12892–1-AP at 1:1000 dilution, actin rabbit antibody, Abcam, ab8227, at 1:1000 dilution, overnight at 4 °C.

Techniques: Expressing, Inhibition, Binding Assay, Staining, Western Blot, Stable Transfection, Immunostaining, Control, Software, Activity 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: Fig. 5. 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 immunofluo- rescence 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 antiSQSTM1/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, 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: Fig. 6. 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 antiSQSTM1/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: Fig. 7. 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 antiSQSTM1/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: Fig. 8. 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 sig- nificance 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 antiSQSTM1/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