Journal: The Journal of Biological Chemistry

Article Title: Chronic treatment with the complex I inhibitor MPP + depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2–associated athanogene 6 (BAG6)

doi: 10.1074/jbc.RA119.010474

Figure Lengend Snippet: List of antibodies used in the study

Article Snippet: BAG5 , Rabbit , NB100-56091 , Novus Biological , 1:1000.

Techniques: Transduction

Journal: bioRxiv

Article Title: Identification of Candidate Mitochondrial Inheritance Determinants Using the Mammalian Cell-Free System

doi: 10.1101/2023.01.23.525177

Figure Lengend Snippet: BAG family molecular chaperone regulator 5 was detected in ejaculated spermatozoa via WB ( A ; predicted mass = 54 kDa) and immunocytochemistry ( B ), where it was detected to localize in the acrosome. After priming, BAG5 still remained localized to what remained of the acrosome ( C ). After 4 and 24 hours of cell-free system exposure, BAG5 was detected in the MS of the treated spermatozoa ( D and E ). BAG5 was detected in the cytoplasm in zygotes 15 hours post structures ( F ) and 25 hours post fertilization, with no obvious association with the fertilizing sperm structures or their remnants ( G ).

Article Snippet: Mouse monoclonal anti-BAG5 (CF810618) was purchased from OriGene Technologies, Rockville, MD.

Techniques: Immunocytochemistry

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: BAG5 is upregulated in DNA damage- and oxidative stress-treated cells but not in hypoxia-treated cells. ( A – C ) U2OS, HeLa, and SH-SY5Y cells were treated with 10 μM of etoposide for 48 h, 250 μM of H 2 O 2 for 24 h, 10 μM of DFX for 48 h, or solvent. RNA was extracted, and BAG5 transcripts were detected by RT-Q-PCR. The relative fold in BAG5 mRNA expression was normalized to the GAPDH control and the solvent control (upper panel). The protein expression of BAG5, p53, HIF-1α, and β-actin was detected by Western blotting (lower panel). The relative fold in protein expression was normalized to that of the internal control β-actin and standardized with the solvent control. Error bars represent the SD of the means calculated using data from three independent experiments (Student’s t-test; * , p < 0.05, ** , p < 0.01, *** , p < 0.001).

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Solvent, Expressing, Control, Western Blot

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: p53 is responsible for BAG5 induction. ( A ) U2OS, HeLa, and SH-SY5Y cells were transfected with various amounts of a p53-expressing plasmid (pcDNA3.1-p53). Expression levels of p53 and BAG5 were detected by Western blotting. β-Actin served as a loading control. After 24 or 48 h of pretreatment with etoposide (10 μM) or H 2 O 2 (250 μM), p53 was repressed in etoposide-treated ( B ) and H 2 O 2 -treated ( C ) U2OS, HeLa, and SH-SY5Y cells by transfection of shp53-3755, shp53-3756, or shLuc control for 48 h and selecting with puromycin (2 μg/ml) for 48 h. The protein expression levels of BAG5, p53, and β-actin were detected by Western blotting. BAG5 transcripts were detected in shLuc and two p53 shRNAs knockdown cells. The expression levels of BAG5 transcripts were normalized to those of GAPDH and standardized with those of shLuc cells. ( D ) Total RNAs and cell lysates were harvested from HCT116 p53 wild-type (p53 +/+ ) and null (p53 -/- ) isogenic colorectal cancer cell lines. BAG5 transcripts were detected by RT-Q-PCR. The relative fold in BAG5 mRNA expression was normalized to the GAPDH control and the vector control (upper panel). The protein expression levels of BAG5, p53, and β-actin were detected by Western blotting (lower panel). The relative fold in protein expression was normalized to the internal control β-actin and standardized with the vector control. Error bars represent the SD of the means calculated using data from three independent experiments (Student’s t-test; *, p < 0.05, **, p < 0.01, ***, p < 0.001).

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Knockdown

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: Transcription factor p53 directly binds to the BAG5 promoter. ( A ) A schematic diagram of human BAG5 promoter (positions −800 to +200 bps). The putative binding sites of the p53 transcription factor were predicted by ALGGEN-PROMO software. U2OS, HeLa and SH-SY5Y cells were treated with 10 μM of etoposide ( B – D ) for 48 h or 250 μM of H 2 O 2 ( E – G ) for 24 h. Cell lysates were harvested post 24- or 48-h treatment, and DNA-protein complexes were immunoprecipitated using an anti-p53 antibody or the irrelevant rabbit IgG control. Q-PCR was performed for DNA-protein complexes to detect the DNA fragments at the BAG5 promoter (from -628 to -608, -364 to -344, -219 to -157, and +2 to +18). GAPDH promoter DNA (from -93 to +64) served as a negative control. The amount of immunoprecipitated DNA in each sample is represented as a signal relative to the total amount of input chromatin.

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Binding Assay, Software, Immunoprecipitation, Control, Negative Control

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: Overexpressed BAG5 may result in loss of its function to promote HSP70 activity. ( A – C ) U2OS, HeLa, and SH-SY5Y cells were transfected with various amounts of a BAG5-expressing plasmid (pCMV-Tag2B-BAG5). Expression levels of BAG5 were detected by Western blotting. β-Actin served as an internal control. ( D – E ) Recombinant GST fusion BAG5 and BAG5 (DARA) protein were purified and stained with Coomassie blue. The HSP70-mediated refolding activity was examined with denatured firefly luciferase in the presence of different concentrations of BAG5 (Student’s t-test; *, p < 0.05, **, p < 0.01).

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Activity Assay, Transfection, Expressing, Plasmid Preparation, Western Blot, Control, Recombinant, Purification, Staining, Luciferase

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: BAG5 is activated upon stresses and colocalized with α-synuclein in the perinuclear compartment. After 24 or 48 h of pretreatment with 10 μM of etoposide or 250 μM of H 2 O 2 , treated SH-SY5Y ( A , C ) or HeLa ( B , D ) cells were paraformaldehyde-fixed and stained using specific BAG5 (green) and α-synuclein (red) antibodies, and DAPI (blue) was used to stain the nuclear DNA. The images (1,260 x) were acquired using an LSM 510 Meta Confocal Microscope (Zeiss). The scale bar shows 20 μm. The percentage of cells with colocalization was determined by counting yellow blobs of at least 1,000 cells (Student’s t-test; *, p < 0.05, ***, p < 0.001).

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Staining, Microscopy

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: BAG5 expression is crucial for α-synuclein aggregation. ( A ) After rotenone treatment (10 μM) for 24 h, images of rotenone-treated α-synuclein (A53T)-expressing SH-SY5Y cells following BAG5 knockdown were captured. Arrows indicate α-synuclein-containing foci. Scale bar, 10 μm. ( B ) Quantified results in ( A ) are shown as the percentage of cells with foci (Student’s t-test; **, p < 0.01). ( C ) Knockdown of BAG5 in ( A ) was displayed by Western blot analysis. ( D ) BAG5 increases SDS-insoluble aggregation of α-synuclein in U2OS, HeLa, and SH-SY5Y cells. α-Synuclein aggregation was detected by the filter-trap assay in cells transfected with various amounts of a BAG5-expressing plasmid. The lysate was diluted in SDS and filtered through nitrocellulose membranes. α-Synuclein immunostaining was detected by the α-synuclein antibody. A representative image and the densitometry data are shown (a.u., arbitrary unit). The values of α-synuclein aggregation were normalized to the amount of aggregation in the empty vector control (Student’s t-test; *, p < 0.05). ( E ) SDS-insoluble α-synuclein aggregation in rotenone-treated and/or α-synuclein (A53T)-expressed SH-SY5Y cells is BAG5-dependent. α-Synuclein aggregation was detected by the filter-trap assay. The values of α-synuclein aggregation were normalized to the amount of aggregation in the vector or solvent control (Student’s t-test; *, p < 0.05).

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Expressing, Knockdown, Western Blot, TRAP Assay, Transfection, Plasmid Preparation, Immunostaining, Control, Solvent

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: BAG5 is increased and colocalized with α-synuclein in SNCA p.A53T mice and PD patients. ( A ) Immunohistochemical staining of substantia nigra sections with a phospho-α-synuclein (Ser129) antibody revealed that the phospho-α-synuclein (Ser129) is expressed mainly in the substantia nigra. The expression of BAG5 (red) and p-α-synuclein (green) was detected mainly at the TH (blue)-positive excitatory synapse in SNCA p.A53T mice. The bars indicate 20 μm (Student’s t-test; ** , p < 0.01). ( B ) Lysates were prepared from human normal control and PD brains. Immunoprecipitations were performed with an anti-BAG5 antibody. Immunoprecipitates were sequentially probed with phospho-α-synuclein (Ser129), anti-α-synuclein, and anti-HSP70 antibodies. Five percent of each lysate used for immunoprecipitation was loaded as input and probed with anti-BAG5, phospho-α-synuclein (Ser129), anti- α-synuclein, and anti-HSP70 antibodies. β-Actin was used as a loading control.

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Immunohistochemical staining, Staining, Expressing, Control, Immunoprecipitation

Journal: Aging (Albany NY)

Article Title: Stress-induced p53 drives BAG5 cochaperone expression to control α-synuclein aggregation in Parkinson's disease

doi: 10.18632/aging.103998

Figure Lengend Snippet: A proposed role of stress- and p53-induced BAG5. A schematic diagram shows that stress induces BAG5 expression through p53 binding at the BAG5 promoter. Under slight stress, a low level of BAG5 improves protein refolding efficiency by acting as a nucleotide exchange factor of HSP70. When the stress is too high, overexpressed BAG5 may accelerate the release of rashly folded clients from the substrate-binding domain (SBD) of HSP70, which causes unfolded protein aggregation.

Article Snippet: The sections were rinsed with PBS, incubated for 30 minutes at room temperature with a blocking solution (BSA in PBS with 0.3% of Triton X-100), and then incubated overnight at 4°C with a rabbit polyclonal antibody raised against TH (Abcam, UK), phospho-Ser129 α-synuclein (Abcam, UK) or BAG5 (Atlas Antibodies, Sweden).

Techniques: Expressing, Binding Assay

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: BAG5 interacts with Akt. ( A ) Schematic representation of Akt and BAG5 structures indicating Bait and Prey and their corresponding interacting domains. ( B ) BAG5 and Akt interact in the yeast two-hybrid system. Akt-KDD (dominant negative mutant of the catalytic domain of Akt) was used as bait to screen a human fetal brain cDNA library in a yeast two-hybrid system. A full-length clone of BAG5 was identified as a novel Akt-interactor protein. The specificity of the interaction between BAG5 and Akt was determined using p53 and pTD1 (T antigen) as negative control bait and prey, respectively, whereas the known interaction between p53 and T antigen served as positive control of the system. All yeast grew on media lacking leucine and tryptophan (-LT, which selects for the presence of the plasmids, left), but only those in which interactions occurred grew in media lacking histidine, leucine, and tryptophan and were positive for the activity of α-galactosidase (-HLT + Xα-Gal, right). ( C , D ) Transfected Akt and BAG5 interact in mammalian cells. HEK293T cells were cotransfected with HA-Akt and Myc-BAG5 ( C ) or with HA-Akt and GST-BAG5 ( D ), as indicated. Total cell lysates (TCL) were either immunoprecipitated using anti-HA antibodies (( C ), IP:HA) or pulled down using glutathione beads (( D ), PD:GST). BAG5 that coimmunoprecipitated with Akt was detected with Myc-specific antibodies ( C ), whereas Akt that interacted with pulled-down BAG5 was detected with HA-specific antibodies ( D ); the expression of both proteins was confirmed in total cell lysates (( C , D ), TCL). ( E ) Schematic representation of BAG5 constructs used to map the interaction with Akt. ( F ) BAG5 linker region (N87-P181) interacts with Akt but not with Erk. The illustrated BAG5 constructs, including the N87-P181 linker region alone or extended towards the first or last three BAG domains fused to GST, were used to identify the region in BAG5 that interacts with Akt, the specificity of the interaction was confirmed using comparison against Erk. GST was used as a negative control. GST-BAG5 pull-down assays were performed in HEK293T cells transiently cotransfected with HA-Akt and HA-Erk and either one of the indicated BAG5 constructs. The ability of either Akt or Erk (both tagged with HA) to interact with BAG5 constructs was revealed by Western blot using anti-HA antibodies. ( G ) BAG5-linker region (N87-P181) interacts with the kinase-dead domain of Akt. The interaction between GST-BAG5-linker region (N87-P181) and EGFP-Akt-KDD was determined using pull-down assays using glutathione resin, the fraction of Akt-KDD that interacted with BAG5-linker region was revealed by Western blot using EGFP antibodies (left panel), whereas its expression was confirmed in total cell lysates (TCL, right panel). ( H ) Endogenous Akt interacts with BAG5. HeLa cell lysates were used to immunoprecipitate endogenous BAG5, and the presence of Akt bound to it was revealed by Western blot in the immunoprecipitate (IP), whereas its expression was confirmed in TCL. As a negative control, ( C ) a non-related antibody (anti-PAK) was used instead of the anti-BAG5 antibody. The arrows indicate the bands corresponding to Akt, BAG5, or IgG heavy chain. The numbers below the top panels ( C , D , F , G , H ) indicate the normalized densitometric value with respect to the condition indicated at 1.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Dominant Negative Mutation, cDNA Library Assay, Negative Control, Positive Control, Activity Assay, Transfection, Immunoprecipitation, Expressing, Construct, Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: BAG5 promotes Akt ubiquitination and degradation. ( A ) BAG5 controls Akt ubiquitination. HeLa cells were transfected with HA-Ubiquitin and Myc-BAG5. Thirty-six hours after transfection, cells were serum-starved overnight and then left untreated or treated with 20 μM of MG132 for 6 h. Akt was immunoprecipitated from total cell lysates, and the presence of ubiquitinated Akt was detected as a high molecular smear in the immunoblots using anti-HA antibodies. ( B ) Akt protein levels are regulated by changes in the expression of BAG5. Increasing amounts of Myc-BAG5 induce a decrease in Akt protein expression, regardless of the effect of MG132, without altering the expression of Erk detected in the same total cell lysates. ( C ) Protein ubiquitination in total cell lysates of HeLa cells transfected with BAG5 and treated with MG132, as indicated in ( A ). ( D ) BAG5 accelerates Akt degradation under Akt destabilizing conditions (ADC). HeLa cells transiently transfected with HA-Akt and Myc-BAG5 were treated with 5 μM of cycloheximide (CHX) and 1 μM of 17-N-Allylamino-17-demethoxygeldanamycin (17AAG) in serum-free media, a condition known to destabilize Akt, during the indicated times. The expression of Akt, phospho-Akt (T-450), and BAG5 was detected by Western blot in total cell lysates (anti-HA for Akt and anti-Myc for BAG5), as well as the expression of Rac, used as loading control. ( E ) BAG5 knockdown attenuates the degradation of Akt under ADC treatment. HeLa cells were transfected with HA-Akt and shRNA-BAG5 for 60 h, followed by ADC treatment for 12 h, as indicated. Akt, BAG5, Erk, and Rac protein levels were analyzed by Western blot with the indicated antibodies. Rac and Erk served as a loading control. The numbers below the top panels ( B , D , E ) indicate the normalized densitometric value with respect to the condition indicated at 1.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Western Blot, Expressing, Control, Knockdown, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Hsp70 mediates Akt degradation promoted by BAG5. ( A ) Hypothetical model showing that BAG5 contributes to Akt regulation by Hsp70. BAG5 structure is shown as predicted by AlphaFold. ( B ) BAG5 interacts with Akt and Hsp70. HEK293T cells were transiently transfected with HA-Akt either in the presence or absence of GST-BAG5 alone or with EGFP-Hsp70, as indicated. GST-BAG5 was isolated by pull-down (PD:GST) from total cell lysates (TCL) using glutathione beds. The interaction of HA-Akt and EGFP-Hsp70 proteins with BAG5 was detected in the pull-down, whereas their expression was confirmed in total cell lysates (TCL), using anti-HA or anti-EGFP antibodies, respectively. ( C ) Hsp70 promotes Akt degradation. HeLa cells transiently transfected with HA-Akt, and increasing amounts of EGFP-Hsp70 were analyzed by Western blot of total cell lysates for the expression of Akt (anti-HA and anti-Akt), Hsp70 (anti-EGFP), and S6, 48 h post-transfection. Anti-S6 immunoblot served as a loading control. ( D ) Graph illustrates the densitometric analysis of HA-Akt expression in the presence or absence of Hsp70 and the effect of BAG5 knockdown on the effect of Hsp70. Bars represent the mean value ± SEM of three independent experiments, **, p < 0.01. ( E ) Hsp70 involves BAG5 to promote Akt degradation. HeLa cells were transiently transfected with shRNA-BAG5 for 24 h; then, cells were transfected again with HA-Akt and EGFP-Hsp70 for 48 h more to achieve 72 h of shRNA BAG5 effect. Akt, Hsp70, BAG5, Erk, and S6 levels were analyzed by immunoblot using antibodies against HA or EGFP for transfected Akt and Hsp70, respectively, or antibodies that recognize endogenous BAG5, Akt, Erk, or S6 as indicated. Erk and S6 immunoblots served as loading controls. The numbers below the top panels ( B , C , E ) indicate the normalized densitometric value with respect to the condition indicated at 1.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Transfection, Isolation, Expressing, Western Blot, Control, Knockdown, shRNA

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Profile of Deubiquitinases and E3 ligases linked to BAG5 expression in BRCA, UCEC, LUSC, and OV TCGA cancer patients. ( A ) BAG5 mRNA expression in BRCA, UCEC, LUSC, and OV TCGA studies in diploid and BAG5 amplified samples. Mean ± S.E.M values. ***, p < 0.001, ****, p < 0.0001. Unpaired t test with Welch’s correction. ( B ) Heatmap for the top ten DUBs correlated genes with BAG5 in BRCA, UCEC, LUSC, and OV cancer studies. ( C ) Heatmap for the top ten E3 ligases correlated genes with BAG5 in BRCA, UCEC, LUSC, and OV cancer studies. ( D ) Akt1 protein expression according to diploid and BAG5 amplified samples. Mean ± S.E.M values. *, p = 0.02, **, p = 0.0048. Unpaired t test with Welch’s correction. ( E ) Highest correlated DUBs with BAG5 in BAG5 amplified samples compared to general correlation. ( F ) Highest correlated E3 ligases with BAG5 in BAG5 amplified samples. Spearman’s correlation coefficients are calculated in cBioPortal. Genes in bold represent DUBs and E3 ligases correlated with BAG5 found in all patients or in BAG5 amplified patients. Underlined genes represent essential DUBs and E3 ligases in breast, uterine, lung, and ovarian cancer cell lines from the CRISPR effect in the DepMap platform.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Expressing, Amplification, CRISPR

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: Phosphorylation of BAG5 by Akt correlates with a reduction in the interaction between them. ( A ) Hypothetical model showing the phosphorylation of BAG5 by Akt. The structure of BAG5 is shown as predicted by AlphaFold, and the indicated phosphorylation site is the most prominent in the phosphoproteomic analysis available at https://www.phosphosite.org/ (accessed on 14 November 2023). ( B ) Akt activation attenuates its interaction with BAG5. HeLa cells transfected with HA-Akt, Myc-BAG5, or both were incubated overnight in serum-free media and then stimulated, or not, with HGF for 15 min, as indicated. Akt was immunoprecipitated from total cell lysates using an anti-HA antibody (IP:HA), and interacting-BAG5 was detected by anti-Myc Western blot. The expression of Akt and BAG5 was confirmed in total cell lysates (TCL) using anti-HA and anti-Myc antibodies, respectively. The effect of HGF on Akt and Erk activation was detected with phospho-specific antibodies. Expression of Erk in total cell lysates served as a loading control. ( C ) Akt degradation induced by BAG5 preferentially occurs under serum starvation conditions. HeLa cells were cotransfected with HA-Akt and increasing amounts of Myc-BAG5. Thirty-six hours post-transfection, cells were incubated with serum-free or serum-supplemented media for an additional 12 h. Expression of Akt, GSK-3β, and BAG5 was analyzed by immunoblot in total cell lysates. GSK-3β served as a loading control. ( D ) Akt phosphorylates BAG5. Cells were transfected with Myc-BAG5 and one of the following Akt constructs: HA-Akt (wild type), myr-Akt (N-myristoylated-Akt, constitutively active), or kinase-negative mutant Akt (Akt-K179M). Twenty-four hours post-transfection, cells that were starved of serum overnight were metabolically labeled with 32 P orthophosphate. BAG5 was isolated by immunoprecipitation with anti-Myc antibodies, and its phosphorylation was detected by autoradiography. ( E ) Schematic representation of BAG5 phosphorylation site at the first BAG domain, which is phylogenetically conserved ( https://www.phosphosite.org/ , accessed on 14 November 2023). The constructs represented at the bottom were used to investigate their potential phosphorylation by Akt. ( F ) Effect of myr-Akt on the phosphorylation of BAG5 and the indicated constructs, corresponding to the first BAG domain and the linker region (as shown in ( E )). Phospho-Akt-substrates were detected with anti-psusAkt antibodies, and isolated constructs were revealed with anti-GST antibodies. The numbers below the top panels ( B – D , F ) indicate the normalized densitometric value with respect to the condition indicated at 1.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Phospho-proteomics, Activation Assay, Transfection, Incubation, Immunoprecipitation, Western Blot, Expressing, Control, Construct, Mutagenesis, Metabolic Labelling, Labeling, Isolation, Autoradiography

Journal: International Journal of Molecular Sciences

Article Title: Akt Is Controlled by Bag5 through a Monoubiquitination to Polyubiquitination Switch

doi: 10.3390/ijms242417531

Figure Lengend Snippet: BAG5 modulates Akt activation and HGF-dependent cell migration. ( A ) BAG5 attenuates the activation of Akt in response to HGF without affecting the activation of Erk. HeLa cells transfected with the control vector, or HA-BAG5, were serum starved and stimulated with increasing amounts of HGF for 15 min. Then, activation of Akt and Erk was detected by Western blot using phospho-specific antibodies, whereas their expression was confirmed using antibodies that detect endogenous proteins. The expression of transfected BAG5 was confirmed using anti-HA antibodies. Graph at the top represents the analysis of Akt phosphorylation determined in three independent experiments. Mean ± S.E.M values. *, p < 0.05. ( B , C ) BAG5 expression reciprocally correlates with Akt activation in conditions in which Akt stability is not affected. ( B ) HeLa cells transiently transfected with increasing amounts of shRNA-BAG5 ( B ) or Myc-BAG5 ( C ) were used to detect Akt activation by Western blot in total cell lysates with a phospho-Akt (Ser473) specific antibody. The expression of total Akt, as well as transfected Myc-BAG5, was confirmed in the same samples, as indicated. The numbers below the top panels ( A – C ) indicate the normalized densitometric value with respect to the condition indicated at 1. ( D ) BAG5 diminishes HGF-dependent chemotactic cell migration. HeLa cells transfected with BAG5 or control plasmid were subjected to chemotaxis assays in Boyden chambers in which they were stimulated with HGF or 10% FBS, as indicated. Graph represents the analysis of relative cell migration determined in three independent experiments. Mean ± S.E.M values. *, p < 0.05. A representative result is shown at the bottom of the graph. ( E ) Model depicting the proposed mechanism of Akt regulation by BAG5. Under starved conditions and BAG5 overexpression, Akt interacts with BAG5, which promotes Akt inhibition and, with the participation of Hsp70, leads to its degradation. When the Akt signaling pathway is stimulated, the interaction between Akt and BAG5 decreases, coincident with Akt-dependent BAG5 phosphorylation.

Article Snippet: Primary antibodies with the following specificity were used: Akt1 (Sigma, St. Louis, MO, USA, P2482), phospho-Akt Thr450 (Cell signaling, catalog 12178), phospho-Akt (Ser473, catalog sc-7985), phospho-Erk1/2 T202/Y204 (Cell Signaling, catalog 9191), S6 ribosomal protein (Cell Signaling, catalog 2217), GSK3β (Cell Signaling, catalog 9323), BAG5 (Imgenex, Odisha, India, IMG-5678), Rac1 (BD-Biosciences, Franklin Lakes, NJ, USA, catalog 610651), HA (Covance, MMS-101R) and GFP (Santa Cruz, sc-9996), Myc (Sigma M4439), Erk2 (Santa Cruz, CA, USA, sc-154) and GST (Santa Cruz, sc-154).

Techniques: Activation Assay, Migration, Transfection, Control, Plasmid Preparation, Western Blot, Expressing, Phospho-proteomics, shRNA, Chemotaxis Assay, Over Expression, Inhibition

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: Kuramochi cells were transfected with anti-miR-4454/BAG5-siRNA/SPARC-siRNA or both anti-miR-4454 and siRNA or scrambled control oligos (Scr). The transfected cells were then used for the functional assays. (A) Migration: Transfected cells were seeded in transwell inserts with 8 μm pores in and allowed to migrate towards medium containing 10% FBS. Migrated cells were fixed, imaged and quantified using image-J software (mean ± SD; 3 independents experiments). (B) Transfected cells were seeded in 96-wella plates and allowed to grow for 5 days. Cell proliferation was determined by MTT assay (mean ± SD; 3 independents experiments). (C) Colony formation: Transfected cells were seeded in 6-well plates and allowed to form colonies. Colonies were fixed after 2–3 weeks, stained (0.005% crystal violet) and counted using image-J software (mean ± SD; 3 independents experiments). * p<0.01, Students t-test. (D) Kaplan–Meier plot for effect of BAG5 (Affy ID:202985_s_at) and SPARC (Affy ID: 212667_at) expression levels on 5-year progression free survival in ovarian cancer patients using KM Plotter. Red: High expression; Black: Low expression.

Article Snippet: Briefly, proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with BAG5 (Santa Cruz Cat# sc-390832), SPARC (Santa Cruz, Cat# sc-73472 or Haematologic Tech,Cat# AON-5031) Cleaved caspase-3 (Cell Signaling, Cat# 9661), cleaved PARP (Cell Signaling, Cat# 5625), phospho-FAK Tyr397(Cell Signaling, Cat# 3283), FAK (Cell Signaling, Cat# 3285) antibodies.

Techniques: Transfection, Control, Functional Assay, Migration, Software, MTT Assay, Staining, Expressing

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) RNAseq was done on Kuramochi and OVCAR4 cells transfected with pre-miR-4454 or scrambled control oligos (n=3/group). Venn diagram showing the number of genes uniquely and commonly downregulated in Kuramochi and OVCAR4 cells overexpressing miR-4454 compared to the respective controls. (B) The 811 common downregulated genes were analyzed for predicted miR-4454 seed matches in their 3’UTR using microRNA target prediction software TargetScan, miRDB and TargetMiner. Heat map showing the extent of downregulation of the 68 genes, with predicted miR-4454 seed matches, in miR-4454 overexpressing Kuramochi and OVCAR4 cells compared to the corresponding controls. (C) qRT-PCR validation of selected targets in OC cells overexpressing miR-4454. qRT-PCR for SPARC, BAG5 and SOX4 was done using RNA from Kuramochi and OVCAR4 cells transfected with pre-miR-4454 or scrambled control oligos. The baseline SOX4 expression was too low and it was not detected. (mean ± SD from 3 independent experiments). * p<0.01, ** p<0.05, Students t-test. (D) 3’UTR luciferase reporter assay for the binding of miR-4454 to the 3’UTR of BAG5 or SPARC. Wild type (WT) or miR-4454 seed match mutated (Mut) BAG5 or SPARC 3’UTR luciferase constructs were co-transfected with pre-miR-4454 (pre-4454) or scrambled control (Scr) in 293T cells, the conditioned medium collected, and the secreted gaussia luciferase and alkaline phosphatase activity was measured. Luciferase activity was normalized to the alkaline phosphatase activity for transfection normalization (mean±SD; 3 independent experiments). (E-F) Functional effects of miR-4454 targets. Kuramochi, OVCAR4 and OVCAR8 cells were transfected with BAG5 siRNA (E) or SPARC siRNA (F) or scrambled control oligo (Scr) and the effects of silencing them were evaluated on the ability of the cells to migrate, proliferate and form colonies. (E) and (F) left panel - Migration: Transfected cells were seeded in transwell inserts with 8 μm pores in and allowed to migrate towards medium containing 10% FBS in the lower chamber. Migrated cells were fixed, imaged and quantified using image-J software (mean ± SD; 3 independents experiments). Representative images of migrated cells are provided over each treatment group. (E) and (F) middle panel - Proliferation: Transfected cells were seeded in 96-well plates and allowed to grow for 5 days. Cell proliferation was determined by MTT assay (mean ± SD; 3 independents experiments). (E) and (F) right panel - Colony formation: Transfected cells were seeded in 6 well plates and allowed to form colonies. Colonies were fixed after 2–3 weeks, stained (0.005% crystal violet) and counted using image-J software (mean ± SD; 3 independents experiments). Representative images of colonies are provided. * p<0.01, Students t-test.

Article Snippet: Briefly, proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with BAG5 (Santa Cruz Cat# sc-390832), SPARC (Santa Cruz, Cat# sc-73472 or Haematologic Tech,Cat# AON-5031) Cleaved caspase-3 (Cell Signaling, Cat# 9661), cleaved PARP (Cell Signaling, Cat# 5625), phospho-FAK Tyr397(Cell Signaling, Cat# 3283), FAK (Cell Signaling, Cat# 3285) antibodies.

Techniques: Transfection, Control, Software, Quantitative RT-PCR, Biomarker Discovery, Expressing, Luciferase, Reporter Assay, Binding Assay, Construct, Activity Assay, Functional Assay, Migration, MTT Assay, Staining

Journal: Molecular cancer research : MCR

Article Title: Signals from the metastatic niche regulate early and advanced ovarian cancer metastasis through miR-4454 downregulation

doi: 10.1158/1541-7786.MCR-19-1162

Figure Lengend Snippet: (A) The OC cells (OVCAR3, OVCAR4 and Kuramochi) were transfected with pre-miR-4454 or scrambled control oligo (Scr) (left panel) or with BAG5 siRNA or scrambled control oligos (Scr) (right panel). Cells were allowed to grow for 48 h and then lysed in RIPA buffer. Proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with BAG5, cleaved PARP, cleaved caspase-3 and p53 antibodies. Representative blots from 3 independent experiments are shown. (B) OVCAR3, OVCAR4 or Kuramochi cells were transfected with pre-miR-4454 or scrambled control oligo (Scr) (left panel) or with SPARC siRNA or scrambled control oligos (Scr) (right panel). Cells were allowed to grow for 48 h and then lysed in RIPA buffer. Proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with SPARC, phosphorylated FAK at Tyr397 (pFAK) and total FAK antibodies. Representative blots from 3 independent experiments are shown. (C) Schematic overview of findings. Paracrine signals from microenvironmental fibroblasts lead to downregulation of miR-4454 in metastasizing OC cells. The downregulation of miR-4454 promotes metastatic colonization through the concomitant increased expression of its targets SPARC and BAG5, which help by activating FAK, promoting mutant p53 gain of function by its stabilization and inhibiting apoptosis respectively.

Article Snippet: Briefly, proteins were separated by 4–20% gradient SDS-PAGE and transferred to nitrocellulose membrane, probed with BAG5 (Santa Cruz Cat# sc-390832), SPARC (Santa Cruz, Cat# sc-73472 or Haematologic Tech,Cat# AON-5031) Cleaved caspase-3 (Cell Signaling, Cat# 9661), cleaved PARP (Cell Signaling, Cat# 5625), phospho-FAK Tyr397(Cell Signaling, Cat# 3283), FAK (Cell Signaling, Cat# 3285) antibodies.

Techniques: Transfection, Control, SDS Page, Membrane, Expressing, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miRNA-770-5p expression is upregulated in patients with type 2 diabetes and miRNA-770-5p knockdown protects pancreatic β-cell function via targeting BAG5 expression

doi: 10.3892/etm.2021.10096

Figure Lengend Snippet: BAG5 is a target gene of miR-770-5p. (A) TargetScan was used to predict the binding sites between BAG5 and miR-770-5p. (B) Transfection efficiency of miR-770-5p mimic. (C) Dual luciferase reporter assays were performed to verify the interaction between BAG5 and miR-770-5p. (D) Reverse transcription-quantitative PCR was performed to detect BAG5 expression levels in the peripheral blood of patients with T2DM and healthy volunteers. ** P<0.01 vs. mimic control; ## P<0.01 vs. healthy. BAG5, Bcl-2 associated athanogene 5; miR, microRNA; T2DM, type 2 diabetes mellitus; WT, wild-type; MUT, mutant; UTR, untranslated region.

Article Snippet: Min6 cells were transfected with 100 nM miR-770-5p inhibitor (5'-UGGCCCUGACACG UGGUACUGGA-3'; Shanghai GenePharma Co., Ltd.), 100 nM inhibitor control (5'-CAGUACUUUUGUGUAGUACAA-3'; Shanghai GeneChem Co., Ltd.), 2 µM control-small interfering (si)RNA (cat. no. sc-37007; Santa Cruz Biotechnology, Inc.), 2 µM BAG5-siRNA (cat. no. sc-72605; Santa Cruz Biotechnology, Inc.) or 100 nM miR-770-5p inhibitor + 2 µM BAG5-siRNA using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Binding Assay, Transfection, Luciferase, Real-time Polymerase Chain Reaction, Expressing, Mutagenesis

Journal: Experimental and Therapeutic Medicine

Article Title: miRNA-770-5p expression is upregulated in patients with type 2 diabetes and miRNA-770-5p knockdown protects pancreatic β-cell function via targeting BAG5 expression

doi: 10.3892/etm.2021.10096

Figure Lengend Snippet: Effect of UA treatment on miR-770-5p and BAG5 expression levels in Min6 cells. Min6 cells were treated with UA (5 mg/dl) for 24 h. (A) miR-770-5p expression levels were detected via RT-qPCR. BAG5 (B) mRNA and (C) protein expression levels were determined via RT-qPCR and western blotting, respectively. ** P<0.01 vs. control group. UA, uric acid; miR, microRNA; BAG5, Bcl-2 associated athanogene 5; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Min6 cells were transfected with 100 nM miR-770-5p inhibitor (5'-UGGCCCUGACACG UGGUACUGGA-3'; Shanghai GenePharma Co., Ltd.), 100 nM inhibitor control (5'-CAGUACUUUUGUGUAGUACAA-3'; Shanghai GeneChem Co., Ltd.), 2 µM control-small interfering (si)RNA (cat. no. sc-37007; Santa Cruz Biotechnology, Inc.), 2 µM BAG5-siRNA (cat. no. sc-72605; Santa Cruz Biotechnology, Inc.) or 100 nM miR-770-5p inhibitor + 2 µM BAG5-siRNA using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

Journal: Experimental and Therapeutic Medicine

Article Title: miRNA-770-5p expression is upregulated in patients with type 2 diabetes and miRNA-770-5p knockdown protects pancreatic β-cell function via targeting BAG5 expression

doi: 10.3892/etm.2021.10096

Figure Lengend Snippet: Regulatory effect of miR-770-5p on BAG5 expression in Min6 cells. Min6 cells were transfected with control-siRNA, BAG5-siRNA, inhibitor control, miR-770-5p inhibitor or BAG5-siRNA + miR-770-5p inhibitor for 48 h. (A) Transfection efficiency of miR-770-5p inhibitor. The transfection efficiency of BAG5-siRNA was assessed via (B) RT-qPCR and (C) western blotting. BAG5 (D) mRNA and (E) protein expression levels were determined via RT-qPCR and western blotting, respectively. ** P<0.01 vs. control; ## P<0.01 vs. inhibitor. miR, microRNA; BAG5, Bcl-2 associated athanogene 5; siRNA, small interfering RNA; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: Min6 cells were transfected with 100 nM miR-770-5p inhibitor (5'-UGGCCCUGACACG UGGUACUGGA-3'; Shanghai GenePharma Co., Ltd.), 100 nM inhibitor control (5'-CAGUACUUUUGUGUAGUACAA-3'; Shanghai GeneChem Co., Ltd.), 2 µM control-small interfering (si)RNA (cat. no. sc-37007; Santa Cruz Biotechnology, Inc.), 2 µM BAG5-siRNA (cat. no. sc-72605; Santa Cruz Biotechnology, Inc.) or 100 nM miR-770-5p inhibitor + 2 µM BAG5-siRNA using Lipofectamine ® 3000 reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Quantitative RT-PCR, Western Blot, Small Interfering RNA, Real-time Polymerase Chain Reaction

Journal: Cell Death Discovery

Article Title: Neuromodulation of BAG co-chaperones by HIV-1 viral proteins and H 2 O 2 : implications for HIV-associated neurological disorders

doi: 10.1038/s41420-021-00424-0

Figure Lengend Snippet: A Immunoblot results indicated H 2 O 2 treatment caused time and dosage-dependent decreases in neuronal BAG3. B qPCR demonstrated H 2 O 2 treatment causes significant decreases in neuronal BAG3 RNA. C In addition to validating H 2 O 2 -induced reductions in BAG3 protein, immunocytochemistry suggested neurons exposed to H 2 O 2 treatment may develop minor dystrophies in their branching processes. D Immunoblotting revealed H 2 O 2 reduced other BAG family members (BAG2, BAG5, BAG6), and exacerbated the reduction in BAG3 observed in response to Tat or Nef. *** p < 0.001. Scale bar equals 50 μM; ×40 magnification. Scale bar equals 20 μM; ×63 magnification.

Article Snippet: The following primary antibodies were used: ANT (Santa Cruz; SC11433), β-Tubulin (Sigma; T8578), Bcl-2 (Sant Cruz; SC7382), BAG1 (Santa Cruz; SC939), BAG2 (Novus Biologicals; NBP159086), BAG3 (Proteintech; 105991AP), BAG5 (Proteintech; 266281AP), BAG 6 (R&D Systems; AF6438), BAX (Santa Cruz; SC493), GAPDH (Santa Cruz; SC32233), Hsc/Hsp 70 (Sant Cruz; SC24), LC3 (Santa Curz; L8918), Nef (Abcam; ab42355), Tat (NIH Aids Reagent Program; R705), VDAC1 (Santa Cruz; SC8017).

Techniques: Western Blot, Immunocytochemistry