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

Journal: Journal of Cell Communication and Signaling

Article Title: miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells

doi: 10.1007/s12079-017-0410-x

Figure Lengend Snippet: In silico target prediction of putative target genes of miR-217

Article Snippet: These primary antibodies comprised rabbit monoclonal anti-integrin alpha V (#60896), anti-protein kinase C iota (PKCι/λ) (#2998), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (#9102) antibodies from Cell Signaling Technology (Cambridge, United Kingdom) and mouse anti-BAG3 (SAB1404732 from Sigma Aldrich).

Techniques: In Silico, Derivative Assay

Journal: Journal of Cell Communication and Signaling

Article Title: miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells

doi: 10.1007/s12079-017-0410-x

Figure Lengend Snippet: Localization of putative miR-217-5p binding sites in selected predicted target genes. Schematic overview of the mRNA composition of PRKCI, BAG3, ITGAV and MAPK1 and with putative miR-217-5p binding sites (blue boxes) in the 3′ untranslated region (3′ UTR) of the respective target gene mRNA (a) and the miRNA:mRNA alignment based on the data in microRNA.org (b)

Article Snippet: These primary antibodies comprised rabbit monoclonal anti-integrin alpha V (#60896), anti-protein kinase C iota (PKCι/λ) (#2998), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (#9102) antibodies from Cell Signaling Technology (Cambridge, United Kingdom) and mouse anti-BAG3 (SAB1404732 from Sigma Aldrich).

Techniques: Binding Assay

Journal: Journal of Cell Communication and Signaling

Article Title: miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells

doi: 10.1007/s12079-017-0410-x

Figure Lengend Snippet: PRKC1, ITGAV, BAG3 and MAPK1 as direct targets for miR-217-5p in HEK293T cells. Relative luciferase activity 3 days after co-transfection of the pMirGLO vector with the binding sites 1 to 4 and the fusion of binding sites 2 and 3 of PRKC1 (a), the binding sites of ITGAV (b) and BAG3 (c) as well with the binding sites MAPK1_1 to 6 and the fusion of binding sites 1–4 (d) with miR-217-5p mimic, miRNA inhibitor anti-miR-217-5p or non-targeting siRNA (NT). Statistical differences between means were tested using unpaired t-test [n = 3 biological and technical replicates; mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001]

Article Snippet: These primary antibodies comprised rabbit monoclonal anti-integrin alpha V (#60896), anti-protein kinase C iota (PKCι/λ) (#2998), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (#9102) antibodies from Cell Signaling Technology (Cambridge, United Kingdom) and mouse anti-BAG3 (SAB1404732 from Sigma Aldrich).

Techniques: Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Binding Assay

Journal: Journal of Cell Communication and Signaling

Article Title: miR-217-5p induces apoptosis by directly targeting PRKCI, BAG3, ITGAV and MAPK1 in colorectal cancer cells

doi: 10.1007/s12079-017-0410-x

Figure Lengend Snippet: Potential pro-apoptotic mechanisms of miR-217-5p regulating the ERK-MAPK pathway at different sites. MiR-217-5p was shown to directly downregulate PRKCI, ITGAV, BAG3 and MAPK1, connected in a signaling network modulating the ERK-MAPK pathway. Binding of extracellular matrix components to integrins comprising a β (ITGB) and α subunit such as αv (ITGAV) may activate the ERK- MAPK signaling pathway via focal adhesion kinase (FAK) activation, growth factor receptor-bound protein 2 (GRB2), and guanine nucleotide exchange factor son of sevenless (SOS), leading to the activation of the kinase cascade including KRAS, BRAF, MEK, and MAPK1 and the activation of, Phosphoinositide 3-kinase (PI3K)/Akt. These pathways culminate in the activation of survival and proliferation promoting transcription factors including c- myc, c-fos or Ets like protein 1 (Elk1), in the induction and stabilization of anti-apoptotic members (Bcl-2, Bcl-xL, Mcl-1) of the Bcl-2 protein family and the inhibition of pro-apoptotic members including the BH3-only members Bad and Bim promoting the initiation of intrinsic apoptosis via Bax and Bak. PRKCI, ITGAV, BAG3 and MAPK1 promote cell survival and proliferation by induction of transcription factors including NK-κB, AP1 or SOX2, or by induction of the ERK-MAPK pathway via Rac1

Article Snippet: These primary antibodies comprised rabbit monoclonal anti-integrin alpha V (#60896), anti-protein kinase C iota (PKCι/λ) (#2998), rabbit polyclonal anti-p44/42 MAPK (Erk1/2) (#9102) antibodies from Cell Signaling Technology (Cambridge, United Kingdom) and mouse anti-BAG3 (SAB1404732 from Sigma Aldrich).

Techniques: Binding Assay, Activation Assay, Inhibition

Journal: Molecular Metabolism

Article Title: Pancreatic beta-cell specific BAG3 knockout results in chronic hyperinsulinemia inducing insulin resistance

doi: 10.1016/j.molmet.2023.101752

Figure Lengend Snippet: Generation and validation of BAG3βKO mouse model . (A) Breeding scheme for generating beta-cell specific BAG3 knockout mice. Only the 4 genotypes of interest are shown in the scheme i) BAG3 f/f : INS1 cre−/− and BAG3 −/− : INS1cre +/+ (Control), ii) BAG3 f/- : Ins1cre +/− and iii) BAG3 f/f : INS1 cre+/+ (BAG3βKO). (B) Schematic image of floxed BAG3 allele and the deletion product after INS1cre recombination; for simplicity, only one allele has been shown. (C) PCR genotyping results of BAG3 floxed mice and INS1cre recombinase mice from DNA obtained from ear punch samples: 1-Wild type (BAG3 −/− and INS1 cre−/− ); 2-heterozygote (BAG3 f/- and Ins1cre +/− ) and 3-Mutant (BAG3 f/f and INS1 cre+/+ ). (D) Representative immunofluorescence images of BAG3 and insulin staining in control and BAG3βKO mice isolated islets. Scale bar: 200 μm. (E) Western blot analysis of pancreatic islets and exocrine tissue to assess BAG3 specific knockout, while (F) no Cre recombination was found in the heart, liver, lung, brain, cerebellum, muscle, and spleen of BAG3βKO mice. (G) The growth curve of the body weight and area under the curve (AUC) monitored over time. (H) Fed and (I) fasting blood glucose levels monitored over time and their AUC. Data are presented as mean ± SEM (n = 6–7 for each group), ∗p < 0.05.

Article Snippet: Next, the cytospin preparations were fixed with 4% paraformaldehyde (Santa Cruz Biotechnology) and incubated with rabbit polyclonal anti-BAG3 (1/200 dilution; NBP2-27398, Novus Biologicals, Milan, Italy) and mouse monoclonal anti-Insulin B (C-12) (1/1000 dilution; sc-377,071, Santa Cruz Biotechnology) antibodies overnight, followed by anti-rabbit Alexa fluor 488 (A-11008, Thermo Fisher Scientific, Waltham, MA, USA) and anti-mouse Alexa fluor 546 (A-11030, Thermo Fisher Scientific, Waltham, MA, USA) secondary antibodies for 1 h at RT.

Techniques: Knock-Out, Control, Mutagenesis, Immunofluorescence, Staining, Isolation, Western Blot