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: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Linear illustration of human BAG3 protein sequence with reported p-sites annotated. (B) Co-IP of FLAG-BAG3 from HEK293 cells revealed significantly enriched proteins, assessed using the rankprod method (n = 4), with a minimum fivefold increase and a P -value ≤ 0.05. Enriched proteins in the sample are marked in respective colors for subcategories. Selected hits are annotated with their gene name. A false discovery rate of 5% is indicated by a dashed line. (C) Enriched phosphatases and respective phosphatase regulators from the BAG3 co-IP sample were assigned to their respective (super-)families of human phosphatases. The phosphoprotein phosphatase family is colored; other Ser/Thr-specific phosphatases are in shades of gray. Gene names are listed next to the corresponding assigned phosphatase identifications. PPMs, metal-dependent protein phosphatases; PSTPs, protein serine/threonine-specific phosphatases. (A, D) Gene ontology enrichment analysis of significantly enriched genes from the BAG3 co-IP sample (n = 277) in (A). The 10 most abundant biological process GO terms are displayed as bars, ranked based on protein counts. Biological processes related to BAG3’s role in protein homeostasis are presented in blue. (E) Gene ontology enrichment analysis of the co-IP sample compared with overall human gene expression, displayed as a heatmap. Fold enrichment was calculated for the BAG3 enriched, not enriched, and total co-IP sets, with overall GO categories annotated at the respective cluster. Statistical significance was determined using Fisher’s test ( P -value < 0.05) with the Bonferroni correction implemented in the PANTHER database. Non-significant changes are colored gray within the heatmap.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Sequencing, Co-Immunoprecipitation Assay, Expressing

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Illustration of the used FLAG-control construct to determine unspecific binding and the N -terminally triple-FLAG–tagged human BAG3 used as the bait for the FLAG co-IP. (B) A schematic overview displays annotated BAG3 domains for orientation. BAG3 sequences available from public databases (377 species, all vertebrates) were aligned by geneious using the MUSCLE algorithm. A heat map representation of the alignment identity score was generated to visualize the sequence conservation of BAG3. Regions in the alignment corresponding to gaps in the reference (BAG3 of homo sapiens ) were excluded. (C) The multiple sequence alignment for the p-site stretches of interest in this study are underlined and represented in more detail using sequence logos (see (C)). (B, C) Phylogenetic analysis of the multiple sequence alignment displayed in (B). A consensus tree was built with Jukes-Contor model and Neighbor-Joining method after 200 bootstrap replications. The consensus tree for the 377 BAG3 orthologs reveals three primary clusters, corresponding to Teleostei, Sarcopterygii, and mammals. Sequence logos display the context of BAG3-pS136 and the p-site cluster (pS284-pS291) phosphorylation sites for these three principal orthologous groups. Phosphorylation sites are not conserved across all vertebrate classes, but they are conserved in mammals. In the tree, the BAG3 positions of mouse, rat, and human are indicated by black triangles, arranged from left to right.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Control, Construct, Binding Assay, Co-Immunoprecipitation Assay, Generated, Sequencing

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) HEK293 cells and A7r5 smooth muscle cells were lysed, and protein levels of BAG3 were assessed in relation to the housekeeping protein GAPDH using immunoblots. Quantitative analysis of BAG3 expression validated elevated expression in A7r5 cells. Statistical significance was evaluated through t tests, incorporating data from eight independent replicates. (B) Alignment of human, mouse, and rat BAG3 orthologs to visualize the conservation of the p-sites of interest. Functional p-site studies were carried out in these three organisms in preliminary studies and in rat and human in this work. (C) Summary of the phosphopeptides used in enzyme kinetic studies for the assessment of PP1c and PP5c-mediated dephosphorylation of BAG3 mimetics. (D) Monitoring of CDC37-pSer13 dephosphorylation in A7r5 lysate upon incubation with PP5, for the indicated incubation time to validate enzymatic activity. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of five independent experiments with adjusted P -values obtained from one-way ANOVA with Sidak correction (*** P = 0.0004), **** P < 0.0001). Mean is shown and error bars represent the SD. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Western Blot, Expressing, Functional Assay, De-Phosphorylation Assay, Incubation, Activity Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) A7r5 smooth muscle cells were treated with increasing concentrations of phosphoprotein phosphatase inhibitors CalA or OA as indicated, or DMSO as control. After 20 min of incubation, cells were lysed, and modulator effects on BAG3-pS136 were determined with immunoblots. The quantification is presented as a bar plot, with the mean depicted with error bars that represent the SEM based on five independent experiments. Statistical significance between inhibitors was determined with t test and P -values are displayed as stars (* P = 0.020, *** P < 0.001). (B, C) Michaelis–Menten kinetic parameters of PP1c were determined against the indicated mono- or bisphosphorylated peptides. Error bars represent the SEM of three independent replicates with technical duplicates. k cat / K m was calculated by comparison with a phosphate standard curve. (D) Overexpressed FLAG-BAG3 from transiently transfected HEK293 cells was single-step affinity immobilized with anti-FLAG beads and treated with PP1c for 30 min. Immunoblot quantification depicts the mean of four independent experiments, the error bar represents the SD, with P -values obtained from a t tests (two-tailed, paired, **** P < 0.001). (E) Monitoring of BAG3-pS136 dephosphorylation in A7r5 lysate upon incubation with PP1c, PP5, or without phosphatase as a control treatment for the indicated incubation time. Endogenous phosphatases were inhibited by the addition of 20 nM CalA. Quantification depicts the results of four independent experiments with P -values obtained from two-way ANOVA with Sidak correction (** P = 0.006 [PP1c], ** P = 0.005 [PP5], **** P < 0.001). Mean is shown and error bars represent the SEM. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Control, Incubation, Western Blot, Comparison, Transfection, Two Tailed Test, De-Phosphorylation Assay

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Illustrative overview of the small molecule and peptide modulator screen to tune PP1 activity in cells. HEK293 cells transiently expressing FLAG-BAG3 were treated as illustrated, lysed, and captured by a single-step affinity enrichment using anti-FLAG beads. Samples were analyzed by immunoblots. Quantification depicts results of three independent experiments with P -values obtained from t tests (two-tailed). Mean is shown with replicates as scatter plot and error bars represent the SD (* P = 0.015, *** P < 0.001). (B) A7r5 smooth muscle cells were incubated with increasing concentrations of Tautomycetin as indicated. Immunoblots of lysate were used to determine the sensitivity of the titrated inhibitor towards BAG3-pS136. Mean is shown as a barplot with replicates as scatter plots, and error bars represent the SD of four independent experiments. Statistical significance between concentrations is determined with t test with Welch’s correction (* P < 0.05). (C) Analysis of phosphorylation-dependent binding of 14-3-3γ to FLAG-BAG3 in A7r5 muscle cells after 20-min modulation of endogenous PP1 before lysis and FLAG-IP enrichment. Results are presented as a barplot, P -values obtained from a t test (*** P = 0.0002, **** P < 0.0001). (D) Immunoblots depicting siRNA-mediated knockdown of all PP1 isoforms combined (PP1α/β/γ) or individual PP1 isoforms (PP1α, PP1β, PP1γ) separately. The displayed blots represent the analyzed protein levels of 24 or 48 h after transfection. (E) Quantification of immunoblotted lysates show level changes of PP1α/β/γ and BAG3 (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Activity Assay, Expressing, Western Blot, Two Tailed Test, Incubation, Binding Assay, Lysis, Knockdown, Transfection

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Schematic illustration of the protein level assessment after treatment of HEK293 cells with pre-designed siRNA. HEK293 cells were transfected with siRNA of PP1 isoforms (PPP1CA/B/C) for 24 and 48 h. (B) Quantification of immunoblotted lysates show phosphorylation level changes of BAG3-pS136 relative to the overall BAG3 levels (n = 3 or 4). Quantification depicts results of three or four independent experiments with P -values obtained from t tests (two-tailed, paired). Error bars represent the SD. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Transfection, Two Tailed Test

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Phosphorylation levels of BAG3-pS136 were compared in HEK293 cells overexpressing FLAG-BAG3 or both FLAG-BAG3 and FLAG-PP5 for 24 h. Immunoblot quantification demonstrates that PP5 overexpression does not affect BAG3-pS136 phosphorylation. Statistical significance was determined through t test (n = 4). (B) Michaelis–Menten kinetic parameters of PP5c were determined against the indicated mono- or bisphosphorylated peptides. Error bars represent the SEM of three independent repeats with technical duplicates. k cat / K m was calculated by comparison with a phosphate standard curve. (C) HEK293 cells overexpressed FLAG-BAG3 and were treated with 1 μM recombinant PP5 after lysis for 1 h (in vitro), or overexpressed FLAG-BAG3 and FLAG-PP5 simultaneously for 24 h (in-cell), or were not treated with phosphatase (Ctrl). FLAG-BAG3 was subjected to affinity enrichment and subsequently evaluated using LC-MS/MS. The theoretical phosphopeptide generated through trypsinization is shown at the top. Targeted phosphoproteomics quantification reveals the complete dephosphorylation of the p-site cluster for both PP5 treatments. Statistical significance was determined through two-way ANOVA (n = 4), error bars represent the SD (**** P < 0.0001, *** P = 0.0002, * P = 0.0021). Indexed retention time (iRT). (D) FLAG-BAG3 mutants mimicking the phosphorylated (4D) and dephosphorylated (4A) state of the BAG3 p-site cluster were overexpressed in HEK293 cells followed by co-IP and MS analysis to assess PP5 binding to each variant compared with WT. Bars represent the mean (n = 3), with significance and error bars calculated using t test (* P = 0.03). (C, E) Samples were subjected to the same treatment as described for (C) to induce dephosphorylation of p-site cluster. This was followed by the analysis of protein quantities bound to BAG3 via immunoblotting, normalized to the amounts bound to the control sample. Statistical significance of changes in protein levels was assessed using data from seven or nine independent replicates, with means presented as bar plots. P -values were calculated using a one-way ANOVA with a 95% confidence interval and analyzed using PRISM/GraphPad version 6 (HspB8: * P = 0.0188; ** P = 0.0002; 14-3-3: P [in vitro] = 0.1668, P [in-cell] = 0.6241). (F) A7r5 cells were transfected with PPP5C siRNA for the specified incubation time and protein levels were determined using immunoblots. Statistical significance in the changes of protein levels was determined based on data from five independent replicates, and the means are presented as bar plots. The P -value was calculated using a two-tailed test with a 95% confidence interval and determined using PRISM/GraphPad version 6 (PP5: *** P = 0.001, ** P = 0.0045, *** P = 0.0006; BAG3: ** P = 0.0097, * P = 0.046, * P = 0.0328). (G) Smooth muscle cells were subjected to treatment with control siRNA or PP5-targeting siRNA (siPP5) followed by treatment with BafA1 to inhibit CASA mediated BAG3 degradation. Statistical significance of changes in protein levels was assessed using data from seven independent replicates, represented as boxplots. P -values were calculated using a two-way ANOVA and analyzed using PRISM/GraphPad version 6. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: Western Blot, Over Expression, Comparison, Recombinant, Lysis, In Vitro, Liquid Chromatography with Mass Spectroscopy, Generated, De-Phosphorylation Assay, Co-Immunoprecipitation Assay, Binding Assay, Variant Assay, Control, Transfection, Incubation, Two Tailed Test

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: (A) Schematic representation of the experimental procedure used for the dephosphorylation analysis of the p-site cluster of FLAG-BAG3 derived from HEK293 cells. Cells were either overexpressing FLAG-PP5 or subjected to in vitro PP5 incubation. This workflow served to decipher the dephosphorylation of the BAG3 p-site cluster (LC-MS/MS; ). (B) HEK293 cells overexpressing FLAG-BAG3 were subjected to various modulations of PP5 activity including PP5 co-overexpression for 24 h, activation using arachidonic acid (2 h, 250 μM), or in vitro incubation with recombinant PP5 (1 h, 1 μM, 30°C) in lysate or on-beads (30 min, 1 μM PP5, 30°C). LC-MS/MS was used to quantitatively assess the dephosphorylation status of individual p-sites within the p-site cluster. (C) Related to . Immunoblots of lysates from BAG3-overexpressing HEK293 cells that were treated with PP5 to ensure equal amounts of proteins were present. (D) Related to . Immunoblots of FLAG-BAG3 co-IP samples from HEK293 cells subjected to dephosphorylation treatments with PP5. (E) A7r5 cells were transfected with PPP5C siRNA for the specified incubation time and protein levels were determined using immunoblots. Statistical significance in the changes of protein levels was determined based on data from five independent replicates, and the means are presented as bar plots. The nonparametric Spearman correlation between BAG3 and PP5 upon PPP5C siRNA treatment verifies the negative correlation between the protein levels. (F) Smooth muscle cells were subjected to treatment with control siRNA or PP5-targeting siRNA (siPP5) followed by treatment with BafA1 to inhibit CASA-mediated degradation. Statistical significance of changes in protein levels of SQSTM1 was assessed using data from seven independent replicates, represented as boxplots. P -values were calculated using a two-way ANOVA and analyzed using PRISM/GraphPad version 6. Source data are available for this figure.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: De-Phosphorylation Assay, Derivative Assay, In Vitro, Incubation, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Over Expression, Activation Assay, Recombinant, Western Blot, Co-Immunoprecipitation Assay, Transfection, Control

Journal: Life Science Alliance

Article Title: Identification of phosphatases that dephosphorylate the co-chaperone BAG3

doi: 10.26508/lsa.202402734

Figure Lengend Snippet: Dephosphorylation of pS136 on BAG3 by PP1 leads to loss of 14-3-3 protein binding. Dephosphorylation of the p-site cluster pS284-pS291 by PP5 enables HspB8 binding, and PP5 depletion increases BAG3 levels in a CASA-dependent manner.

Article Snippet: The inclusion list contained m/z values and retention time information of 14 Bag3 peptides and 9 phosphopeptides as well as the 11 iRT standard peptides (Biognosys).

Techniques: De-Phosphorylation Assay, Protein Binding, Binding Assay

Journal: International journal of molecular sciences

Article Title: 2'-Hydroxycinnamaldehyde, a Natural Product from Cinnamon, Alleviates Ischemia/Reperfusion-Induced Microvascular Dysfunction and Oxidative Damage in Rats by Upregulating Cytosolic BAG3 and Nrf2/HO-1.

doi: 10.3390/ijms252312962

Figure Lengend Snippet: Figure 1. Effect of HCA pretreatment on H2O2-induced H9c2 cell death and BAG3 expression in H9c2 cells. HCA pretreatment (A) but not co-treatment (B) significantly inhibited H2O2-induced H9c2 cell death at a dose of 0.001–0.1 mg/mL. HCA pretreatment induced a dose-dependent upregulation of BAG3 expression in H9c2 cells (C–E). All data are expressed as mean ± SEM (n = 3). * indicates significance vs. CON group (* p < 0.05; ** p < 0.01). # indicates significance vs. H2O2 group (# p < 0.05; ## p < 0.01).

Article Snippet: The primary antibodies used were CD45 (ab10558, Abcam, 1:200), BAG3 (1:1000, Proteintech) [35], Beclin-1 (1:1000, Proteintech) [35], and LC3II (1:1000, Proteintech) [35].

Techniques: Expressing

Journal: International journal of molecular sciences

Article Title: 2'-Hydroxycinnamaldehyde, a Natural Product from Cinnamon, Alleviates Ischemia/Reperfusion-Induced Microvascular Dysfunction and Oxidative Damage in Rats by Upregulating Cytosolic BAG3 and Nrf2/HO-1.

doi: 10.3390/ijms252312962

Figure Lengend Snippet: Figure 2. Effect of HCA preconditioning on mitochondrial and cytosolic BAG3 expression in sham, HCA, I/R, and HCA+I/R groups. (A) Original graphs of cytosolic and mitochondrial BAG3 expres- sion in sham and HCA hearts, determined via Western blot assay. Translocation of mitochondrial BAG3 (B) to cytosolic BAG3 (C) and translocation of mitochondrial cytochrome C (D) are noted between sham and HCA hearts. (E) Original traces of mitochondrial and cytosolic BAG3 expression. Significantly reduced cytosolic BAG3 expression is observed in I/R group vs. sham group, whereas significantly preserved cytosolic BAG3 is observed in HCA+I/R group vs. I/R group (G). Mitochon- drial fractions of BAG3 expression (F) and cytochrome C expression (H) are not significantly different among sham, I/R, and HCA+I/R groups. Each symbol represents the respective individual. All data are expressed as mean ± SEM (n = 3–5). * indicates significance vs. sham group (* p < 0.05; ** p < 0.01). # indicates significance vs. I/R group (# p < 0.05).

Article Snippet: The primary antibodies used were CD45 (ab10558, Abcam, 1:200), BAG3 (1:1000, Proteintech) [35], Beclin-1 (1:1000, Proteintech) [35], and LC3II (1:1000, Proteintech) [35].

Techniques: Expressing, Western Blot, Translocation Assay

Journal: International journal of molecular sciences

Article Title: 2'-Hydroxycinnamaldehyde, a Natural Product from Cinnamon, Alleviates Ischemia/Reperfusion-Induced Microvascular Dysfunction and Oxidative Damage in Rats by Upregulating Cytosolic BAG3 and Nrf2/HO-1.

doi: 10.3390/ijms252312962

Figure Lengend Snippet: Figure 7. Effect of HCA preconditioning on cardiac I/R-induced infarct size, BAG3, and autophagy expression among the three groups. The typical infarct areas of I-VI sections among the three groups are displayed in (A). Percentages of six sections of infarct area in I/R and HCA+I/R groups are indicated in (B). Total infarct area (C), infarct area/AAR (D), and AAR/LV (E) are demonstrated. Cardiac BAG3 expression (F,G), Beclin-1 (H,J), and LC3II (H,K) examined via Western blot analysis are also shown. Immunohistochemistry of BAG3, Beclin-1, and LC3II is shown in (I). Statistical analysis of BAG3, Beclin-1, and LC3II in the three groups is shown in (L–N), respectively. All data are expressed as mean ± SEM (n = 3). * indicates significant differences vs. sham group (** p < 0.01; *** p < 0.001; **** p < 0.0001). # indicates significant differences vs. I/R group (# p < 0.05; ### p < 0.001; #### p < 0.0001).

Article Snippet: The primary antibodies used were CD45 (ab10558, Abcam, 1:200), BAG3 (1:1000, Proteintech) [35], Beclin-1 (1:1000, Proteintech) [35], and LC3II (1:1000, Proteintech) [35].

Techniques: Expressing, Western Blot, Immunohistochemistry