Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: a, Schematic for the surface immunoprecipitation (IP) of BCL2 family proteins. Anti-apoptotic proteins (BCL2, BCLxL, MCL1) in cell extracts were lysed with mild detergent and immobilized onto the surface of the reaction chamber. b, Schematic of sandwich-type immunoassay selectively detecting PPI complex (left) and the total level of surface bait (right). The fluorescence signals from detection antibodies were imaged by a single-molecule co-IP platform. c, Comparing the formation of BCL2-BIM BH3 PPI complex in an intracellular environment by BCL2-mCherry/BIM BH3 -eGFP co-transfection (red) and in vitro mixing of two individual extracts expressing BCL2-mCherry and BIM BH3 -eGFP (black). d, Relative changes of active BAX level from the STS treated HL60 cells after lysed with different detergent types. The monoclonal antibody 6A7 was used to detect the activated BAX . Spurious BAX activation by Triton X-100 was indicated (blue). e, Surface-immobilization of the matching IP antibodies immunoprecipitate BCL2-eGFP from the crude extract with high specificity. f, Comparison of signal to noise ratio (SNR) from single molecule counting analysis (red) and total fluorescence signal integrating analysis (black). The raw data to calculate the SNRs are described in Extended Data Fig. 2. g, Counts of endogenous BCL2-related PPI complexes (BCL2-BIM, BCL2-BAX) from the 30,000 HL60 cells by using immunoassay. Error bars represent mean ± s.d. from independent inter-chip measurement ( n ≥ 2). (Two-sided two-sample t -test, P =1.46x10 - , 1.11x10 - respectively). h , Counts of endogenous BCL2-BIM PPI complexes and inter-chip CVs from the fixed numbers of HL60 cells ( n ≥ 2). i , Inter-chip CVs obtained from independent inter-chip measurement for all the immunoassays and cell numbers ( n ≥ 2).

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Immunoprecipitation, Fluorescence, Co-Immunoprecipitation Assay, Cotransfection, In Vitro, Expressing, Activation Assay, Single Molecule Counting

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: Changes of endogenous PPI profiles of HL60 cells through the apoptosis progression by 2 μM of STS. (a) Active BAX/BAK level, (b) Total levels of anti-apoptotic proteins (BCL2, BCLxL, MCL1), (c) BIM complexes with anti-apoptotic proteins, (d) BAX complexes with anti-apoptotic proteins. e , Schematic for the PPI probe binding assay (PBA) for measuring unoccupied populations of surface-immobilized anti-apoptotic proteins. f, Changes of BIM BH3 PBAs for anti- apoptotic proteins from HL60 cells through the apoptosis progression by STS. g, Schematic for the comparison of the BCL2 PPI complex compositions in different AML cell lines. The density of surface-immobilized BCL2 was constantly maintained by optimizing the total protein concentration of crude cell extracts for all AML cell lines (HL60, THP-1, NB4, U937). h, Compositions of the BCL2 complexes (BIM, BAX, BAD, BAK) in four different AML cell lines. The labeling efficiencies of the immunoassays were applied. i, BCL2-BIM BH3 PBA counts of four AML cell lines. j, The PPI complex compositions of BCL2 in HL60, NB4, and U937 cell extracts. k, Schematic of binding competition between two PPI probes, BIM BH3 -eGFP and BAD- mCherry, on surface immobilized BCL2 from HL60 cells. l, Changes of BCL2-BIM BH3 PBA and BCL2-BAD PBA with increasing amounts of BAD probe. BIM BH3 PPI probe was presented in 10 nM.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Binding Assay, Protein Concentration, Labeling

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: Changes of BCL2-related PPI profile in HL60 cells after ABT-199 (100 nM, 24 hours) treatment. (a) Total levels of anti-apoptotic proteins, (b) Active BAX/BAK level, (c) Total levels of BAX/BAK, (d) BCL2 complexes (BIM, BAX), (e) BCL2-BIM BH3 PBA. f-h, Changes of PPI profile of HL60 cells after higher concentration of ABT-199 (300 nM) treatment. (f) BCL2 complexes (BIM, BAX), (g) Active BAX/BAK level, (h) Total levels of BAX/BAK. i, Schematic model of mode of action of ABT-199. j-l , Changes of MCL1-related PPI profile of U937 cells after AZD-5991 (100 nM, 24 hours) treatment. (j) Active BAX/BAK level, (k) MCL1 complexes (BIM, BAK), (l) MCL1-BIM BH3 PBA. The decrease of MCL1-BAK complex after AZD-5991 treatment was indicated (blue). m, Changes of BCLxL complexes (BIM, BAX, BAD, BAK) from U937 cells after AZD-5991 (100 nM, 24 hours) treatment. The increase of BCLxL-BAK complex after AZD-5991 treatment was indicated (green). n, Comparison of relative changes of BAK complexes from U937 cells after AZD-5991 treatment. The relative changes were obtained from the indicated data in (k) and (m). o, Schematic model of mode of action of AZD-5991. (d, k, m) The labeling efficiencies of the immunoassays were applied.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Predicting the efficacy of BH3 mimetics through the profiling of multiple protein complexes

doi: 10.1101/2023.01.16.524337

Figure Lengend Snippet: a, Schematic for generating linear regression correlation between ex vivo efficacy of ABT-199 and PPI profiles from primary AML samples. The collected AML cells were cultured and treated with 0-1 μM of ABT-199, and the area under curves (AUCs) of cell viability were obtained as ex vivo drug efficacy (upper). ∼1.2x10 of primary AML cells on the same cohort were performed single-molecule co-IP profiling (lower) ( n =32). b , Pearson’s correlations between ex vivo AUC of ABT-199 and PPI profiles for primary AML biopsies. c, d , Correlations between ex vivo AUC and single BCL2-related PPI metrics. (c) BCL2-BAX complex, (d) BCL2- BIM complex. e, Correlation between ex vivo AUC and combination of multiple PPI metrics (BCL2-BIM BH3 PBA, BCL2-BAX CPX, and BCLxL-BAK CPX). The statistical indicators (coefficient, p -value) of each metric were displayed. f, Clinical features and the ABT-199 administration history of BC-7064. g, Comparison of the PPI profiles from the initial and the relapsed BC-7064 samples. h, The estimated scores of BC-7064 and BC-7064-R from model (e). i, Investigating the change of the estimated score and ex vivo AUC depending on the relapse of the BC-7064.

Article Snippet: All BCL2 family proteins ( BCL2 (HG10195-M; SinoBiological), BCLxL (HG10455-M; SinoBiological), MCL1 (HG10240-M; SinoBiological), BIM EL (HG13816-G; SinoBiological), BAD (HG10020-M; SinoBiological), BAX (HG11619-M; SinoBiological), BAK (HG10450-M; SinoBiological) and NOXA (HG16548-U; SinoBiological)) were isolated from their respective human cDNA.

Techniques: Ex Vivo, Cell Culture, Co-Immunoprecipitation Assay

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Molecular interactions among tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), Beclin 1, and ubiquitin-specific protease 14 (USP14). (A) Truncated mutants of Beclin 1. Myc-tagged Beclin 1 truncated mutants were generated as described in Section “ .” BH3, Bcl-2 Homology 3; CCD, coiled coil domain; ECD, evolutionarily conserved domain. (B) Expression vectors of Myc-tagged Beclin 1 wild type (wt) and truncated mutants were co-transfected with Flag-tagged USP14 plasmid into HEK293T cells followed by immunoprecipitation (IP) and western blotting analyses. (C) Myc-tagged Beclin 1 wt, Myc-tagged Beclin 1 truncated mutants, and mock as control plasmid were co-transfected with Flag-tagged TRAF6 into HEK293T cells followed by IP and western blotting analyses. (D) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged Beclin 1 into HEK293T cells followed by IP and western blotting analyses. (E) Flag-tagged TRAF6 wt, Flag-tagged TRAF6 truncated mutants, and mock as control plasmid were co-transfected with Myc-tagged USP14 into HEK293T cells followed by IP and western blotting analyses. (F) A schematic model showing molecular interactions among TRAF6, Beclin 1, and USP14. TRAF-C, C-terminal TRAF domain.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Generated, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) inhibits ubiquitination of Beclin 1. (A) Myc-tagged Beclin 1, Flag-tagged tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), and mock as control plasmid were co-transfected with Flag-tagged USP14 plasmid at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to immuno-blotting (IB) assay using anti-USP14, anti-TRAF6, anti-Flag, or anti-Myc antibody. *The intensity of Flag-USP14 was measured by ImageJ. Fold change relative to Flag-USP14 of lane 3 was calculated. **The intensity of Flag-TRAF6 was measured by ImageJ. Fold change relative to Flag-TRAF6 of lane 2 was calculated. (B) Myc-tagged Beclin 1, HA-tagged Ub, Flag-tagged TRAF6, and mock as control plasmid were co-transfected with Flag-tagged USP14 vector at different concentrations. At 38 h post-transfection, transfected cells were extracted, immunoprecipitated with anti-Myc antibody, and subjected to IB assay using anti-HA, anti-Myc, anti-USP14, or anti-TRAF6 antibody. (C) A schematic model showing the inhibition of Beclin 1 ubiquitination by USP14.

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Inhibition

Journal: Frontiers in Immunology

Article Title: Ubiquitin-Specific Protease 14 Negatively Regulates Toll-Like Receptor 4-Mediated Signaling and Autophagy Induction by Inhibiting Ubiquitination of TAK1-Binding Protein 2 and Beclin 1

doi: 10.3389/fimmu.2017.01827

Figure Lengend Snippet: Ubiquitin-specific protease 14 (USP14) negatively regulates autophagy induction and toll-like receptor 4 (TLR4)-mediated signaling. (A) Upon TLR4 stimulation, tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) interacts with Beclin 1 through its coiled coil (CC) domain, induces ubiquitination of Beclin 1, and facilitate autophagy induction (upper). In contrast, USP14 competitively interacts with Beclin 1 to the CC domain of TRAF6. This leads to the suppression of Beclin 1 ubiquitination by TRAF6, resulting in the inhibition of autophagy induction (down). (B) Following TLR4 stimulation, ubiquitinated TRAF6 is associated with the TAB 2–TAK1–TAB 1 complex through an interaction between its polyubiquitinated chain and TAB 2. TAB 2 is then ubiquitinated and the complex facilitates the activation of TAK1. Simultaneously, the IKK complex is associated with the former complex through the polyubiquitinated chain and TAB 2, leading to the activations of nuclear factor-kappa B (NF-κB) and p38 (left). In contrast, the interaction between TAB 2 and USP14 may lead to the deubiquitination of TAB 2, thus inhibiting the association of IKKs, which inhibits the activation of NF-κB and p38 (right).

Article Snippet: Flag-tagged Beclin 1 vector was purchased from Addgene (Cambridge, MA, USA).

Techniques: Inhibition, Activation Assay

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: BAG3 is phosphorylated at T285 during mitosis. ( A ) Schematic representation of BAG3 modular domains and phosphorylated residues, as identified by AP-MS. See also for MS data. Sequence alignments show the interspecies conservation of the identified phosphorylated motif. BAG3-GFP DMB and BAG3-GFP QMB constructs were generated by alanine substitution of designated residues (box). ( B ) Western blots of BAG3-GFP IPs prepared from HeLa-RFP-H2B cells growing asynchronously (AS) or arrested in mitosis by nocodazole treatment (M) and recovered by a mitotic shake-off, showing the mitotic mobility shift of BAG3-GFP constructs. Cells were transfected with BAG3-specific siRNA and transduced with Ad-BAG3-GFP. ( C ) Representative Western blots of HeLa cell extracts that have been transfected with control siRNA or BAG3-specific siRNA targeting BAG3 open reading frame (siBAG3 [ORF]), showing the pT285-BAG3 antibody specificity; M: nocodazole-arrested cells recovered by a mitotic shake-off; AS: asynchronous cells; levels of total BAG3 and GAPDH (loading control) are shown. ( D ) BAG3-GFP IPs were prepared from asynchronous (AS) or from mitotic HeLa-RFP-H2B cells synchronized by nocodazole and recovered by mitotic shake-off after transduction of cells with Ad-GFP-BAG3, as indicated; Western blots show total levels of GFP-BAG3 proteins and pT285-BAG3. ( E ) Extracts were prepared from asynchronous (AS) or from mitotic HeLa-RFP-H2B cells synchronized in early mitosis by nocodazole or arrested at the G2/M stage by RO3306. Western blots of endogenous proteins were performed as indicated; GAPDH: loading control. ( F ) Schematic of the protocol used. HeLa-RFP-H2B cells synchronized in mitosis by nocodazole, were recovered by a mitotic shake-off, and then seeded on poly-L-lysine-coated dishes in fresh medium, with or without MG132 (5 µM) to inhibit mitotic exit. ( G ) Cells were collected at different times and extracts were analyzed by Western blots, as indicated; GAPDH: loading control.

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Protein-Protein interactions, Sequencing, Construct, Generated, Western Blot, Mobility Shift, Transfection, Transduction, Control

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: CDK1 regulates BAG3 phosphorylation dynamic at T285. ( A ) Schematic of the protocol used. Extracts were prepared from mitotic HeLa-RFP-H2B incubated for 1 h in the presence of chemical inhibitors of the following kinases: RO3306 (CDK1, 8 µM), Purvanolol A (CDKs, 10 µM), MLN8237 (Aurora A, 1 µM), and BI2536 (PLK1, 1 µM) or the vehicle only (DMSO); mitotic cells were collected by a mitotic shake-off. Immunoblotting was performed as indicated; GAPDH: loading control. See also . ( B ) Autoradiogram showing phosphorylation of recombinant GST-BAG3 by a purified CDK1-cyclin B1 complex in the presence of 32 P-ATP; positive control: Histone H1; negative control: GST. ( C ) Immunoblotting of in vitro phosphorylated BAG3 using the phospho-specific pT285-BAG3 antibody; total levels of GST-BAG3 are shown. ( D ) BAG3-GFP IPs were prepared from asynchronous (AS) or mitotic HeLa-Flp-In T-REx- BAG3-GFP WT (nocodazole-arrested) transfected with BAG3-specific siRNA (siBAG3 [3′UTR_1], 48 h) and treated with doxycycline to induce BAG3-GFP expression (1 ng/mL, 16 h). Western blots of the GFP IPs were performed as indicated; levels of CDK1, pY15-CDK1 (inactive CDK1), p62, and BAG3-GFP in total cell extracts are shown (Input).

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Phospho-proteomics, Incubation, Western Blot, Control, Recombinant, Purification, Positive Control, Negative Control, In Vitro, Transfection, Expressing

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: Non-phosphorylatable BAG3-GFP DMB expression in BAG3-depleted cells, as phospho-mimetic BAG3-GFP T285D , cannot rescue spindle dynamics and mitotic rounding defects. ( A ) Depletion-rescue experiments were performed in HeLa-RFP-H2B transfected with siBAG3 (3’UTR_1) or control siRNA and transduced with recombinant adenoviruses driving expression of BAG3-GFP proteins. Cells were synchronized in mitosis by a double thymidine block. ( B ) Total cell extracts were analyzed by Western blots using anti-BAG3 and anti-GAPDH; levels of endogenous BAG3 depletion (>75% reduction) and exogenous BAG3-GFP levels were estimated by loading increasing amounts of control cell extract (siCtl, 1/8, 1/4, 1/2, 1), or from an extract of BAG3-depleted cells transduced with Ad-BAG3-GFP, respectively. ( C ) Representative spinning disk confocal time-lapse sequences of cells from ( B ) transduced with BacMam-RFP-α-tubulin, showing normalization of spindle dynamics in BAG3-depleted cells upon reintroduction of BAG3-GFP WT , but not by non-phosphorylatable BAG3-GFP DMB expression or phosphomimetic BAG3-GFP T285D . The green asterisks designate a spindle pole showing normal dynamic in a BAG3-depleted cells expressing wild-type BAG3-GFP, while magenta asterisks designate a spindle pole showing abnormal motility in a BAG3-depleted cell expressing BAG3-GFP DMB ; Bar: 10 μm. The graph shows quantification of percentages of rounded cells with spindle dynamic defects defined as spindle rocking or stalled in mitosis ± spindle rocking; means ± SE of 92 to 525 cells from at least 3 independent experiments. Statistical significance was analyzed with the Fisher Exact Test ****: p < 0.0001. See also for representative phenotypes. ( D ) Epifluorescence images showing the representative phenotype of a rounded mitotic cell at metaphase (siCtl) compared to a flat mitotic cell at metaphase (siBAG3 [3′UTR_2]); F-actin, tubulin, and DNA were stained using phalloidin (green), α-tubulin antibody (magenta) and Hoechst (white), Bar: 10 μm. The graph depicts percentages of cells with mitotic cell rounding defects; means ± SE of 600 to 622 cells from 3 independent experiments. Statistical significance was analyzed by the Fisher Exact Test ****: p < 0.0001; ns, not significant. See also for Western blots of BAG3 depletion levels and for impacts of BAG3-GFP constructs on the aggresomal targeting of ubiquitinated proteins.

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Expressing, Transfection, Control, Transduction, Recombinant, Blocking Assay, Western Blot, Staining, Construct

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: BAG3 phosphorylation at T285 is regulated by p62 during mitosis. ( A ) Western blots of extracts from asynchronous (AS) or mitotic HeLa cells (M) synchronized in mitosis by nocodazole and collected by mitotic shake-off. Cells were transfected with control siRNA (siCtl) or p62-specific siRNAs (sip62) and levels of pT285-BAG3, BAG3, p62, and GAPDH (loading control) are shown. Graph depicting the fold change in pT285-BAG3 levels, as estimated relative to cells transfected with control siRNA and normalized relative to GAPDH levels; means ± SE from 3 independent experiments. Statistical significance was analyzed by the Student′s t -Test ****: p < 0.0001. See also that depicts the fold change in BAG3 total levels as estimated relative to cells transfected with control siRNA and normalized relative to GAPDH levels. ( B ) Western blots of cell extracts prepared from asynchronous (AS) or from mitotic HeLa cells (M) synchronized in mitosis by nocodazole and collected by mitotic shake-off; cells were transfected with control siRNA (siCtl) or BAG3-specific siRNA (siBAG3 [ORF]) or HSPB8-specific siRNAs, and protein levels are shown as indicated. Graphs depicting the fold change in total p62 levels (p62) normalized relative to GAPDH levels, and the fold change in pT269-S272-p62 levels (p-p62) normalized relative p62 total levels, as estimated relative to cells transfected with control siRNA. Data are means ± SE from 3 independent experiments. ( C ) Scheme recapitulating putative functional relationships: CDK1 phosphorylates p62 that in turn enhances CDK1 activity ; the increased mitotic association between the BAG3-HSPB8 chaperone complex and p62 may promote BAG3 phosphorylation and stabilize p62 during mitosis.

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Phospho-proteomics, Western Blot, Transfection, Control, Functional Assay, Activity Assay

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: The sequestosome p62 forms mitotic inclusion bodies (MIBS) enriched in CDK1-phosphorylated p62 and K63 polyubiquitin chains. ( A ) Representative single-plane confocal images of HeLa cells synchronized in mitosis by a double thymidine block, showing staining of p62, K63 polyubiquitin (K63-Ub), and DNA (Hoechst) in cells at different mitotic stages compared to a cell in interphase; M: metaphase; A: anaphase; I: interphase. Bar: 10 μm. ( B ) Deconvolved single-plane confocal images of a representative HeLa cell at metaphase from a non-synchronized cell population, showing staining of pT269-S272-p62 (CDK1-induced), pS403-p62 (stress-induced), and total p62. Enlarged views of the boxed regions emphasize MIBS spherical shape and enrichment in p62 phosphorylated at mitotic sites (T269-S272) but not at the canonical stress-induced site (S403); Bars: 10 μm or 2.5 µm. ( C ) Quantification of polyubiquitin clusters in non-synchronized HeLa cells at prometaphase-metaphase. Cells were transfected with control siRNA or p62-specific siRNAs and stained using anti-α-tubulin and anti-ubiquitin antibodies, means ± SE from 102 to 136 cells from at least 4 independent experiments. Statistical significance was analyzed using the Kruskal–Wallis test and Dunn′s multiple comparisons: ****, p < 0.0001. See also for the representative phenotype in p62-depleted cells and for MIBS staining in other cell types. ( D ) p62 or control IPs were prepared using anti-p62 or rabbit IgG, respectively, from mitotic HeLa cells (nocodazole-treated and recovered by a mitotic shake-off); cells were treated with a reversible crosslinker (DTME) or the vehicle prior to cell lysis and p62 IPs were analyzed by Western blot using anti-p62, anti-BAG3, and anti-K63 polyubiquitin.

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Blocking Assay, Staining, Transfection, Control, Ubiquitin Proteomics, Lysis, Western Blot

Journal: Cells

Article Title: CDK1-Mediated Phosphorylation of BAG3 Promotes Mitotic Cell Shape Remodeling and the Molecular Assembly of Mitotic p62 Bodies

doi: 10.3390/cells10102638

Figure Lengend Snippet: The molecular assembly of p62-MIBS depends on BAG3 phosphorylation and HSPB8. ( A ) Quantification of MIBS number per cell (prometaphase-metaphase) in HeLa cells transfected with control siRNA (siCtl) or BAG3-specific siRNAs or HSPB8-specific siRNAs, and stained for endogenous p62, α-tubulin, and DNA; means ± SE from 70 to 136 cells from at least 3 independent experiments. Statistical significance was analyzed with the Kruskal–Wallis test and Dunn′s multiple comparisons: ****, p < 0,0001. ( B ) Schematic of the protocol used for depletion-rescue analyses of MIBS assembly in Flp-In T-Rex HeLa cell lines. Western blots of extracts from cells treated with BAG3-specific siRNA (3′UTR_2) and doxycycline (1 ng/mL, 16 h), showing levels of BAG3-GFP WT , BAG3-GFP DMB , and BAG3-GFP T285D as compared to endogenous BAG3 levels, as estimated by loading increasing amounts of control Flp-In T-Rex HeLa cell extract (1, 1/4, 1/2, 1/8); HSPB8 and GAPDH levels (loading controls) are shown. ( C ) Maximum intensity projections of confocal image stacks from mitotic Flp-In T-Rex HeLa cell lines expressing GFP as a control or the indicated BAG3-GFP constructs; cells were treated as in (B) and stained for p62, DNA (Hoechst), and β-tubulin (spindle, not shown). A heatmap pseudo-color intensity scale was applied to the p62 staining channel to emphasize impacts on MIBS number and size; Bar: 10 μm. Cells were synchronized in mitosis with a double thymidine block and BAG3-GFP proteins were induced with doxycycline (1 ng/mL, 16 h) during the first thymidine block. The graph shows MIBS number per cell, as quantified in cells at prometaphase-metaphase, means ± SE from 50 to 86 cells from 2 representative experiments. Statistical significance was analyzed with the Kruskal–Wallis test: ****, p < 0.0001; *, p < 0.05.

Article Snippet: Ad-BAG3-GFP S284A-T285A (DMB), Ad-BAG3-GFP S284A-T285A-S289A-S291A (QMB), and Ad-BAG3-GFP T285D were produced by Welgen (Worcester, MA, USA), by subcloning the BAG3-GFP constructs into Adenovirus type C strain 5 vector lacking the E1 and E3 regions.

Techniques: Phospho-proteomics, Transfection, Control, Staining, Western Blot, Expressing, Construct, Blocking Assay

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: Cell lysates from rat neurons transduced with lentivirus expressing scrambled or shBAG3 shRNA at DIV16 were collected at DIV 22. MAP6 and clathrin were selected from the list of proteins that co-immunoprecipitated with BAG3 that were identified by mass spectrometry for verification. (a) Cell lysates were immunoprecipitated with an anti-BAG3 antibody and probed for the presence of MAP6. (b) Cell lysates were immunoprecipitated with an anti-BAG3 antibody and probed for the presence of clathrin. The same amount of rabbit IgG was used to verify the specificity of the immunoprecipitation. A fraction of cell lysate was used as input control. GAPDH is used as a loading control. the positions at which molecular weight markers (kDa) migrated are indicated at the left.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transduction, Expressing, shRNA, Immunoprecipitation, Mass Spectrometry, Control, Molecular Weight

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: Rat cortical neurons were transduced with lentivirus expressing scrambled (Scr) or BAG3 shRNA. Corresponding lysates were immunoprecipitated for BAG3 and associating proteins were run through LC-MS/MS. (A) KEGG enrichment analysis of BAG3 associated proteins with PSM ratios greater than 3 (Scr/shBAG3), FDR-adj. p-value < 0.1. (B) BAG3-associated protein interaction map using STRING. Line thickness indicates the literature interaction. Nodes directly linked to vacuolar processes are labeled in red, and nodes that are related to chaperone/co-chaperone proteins are labeled in green.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transduction, Expressing, shRNA, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Labeling

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: STRING analysis reveals that BAG3 associated with a diverse set of proteins which influence on endocytosis. STRING analysis reveals that BAG3 associated with a diverse set of proteins which influence on endocytosis pathway. Magnified image of .

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques:

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A) Rat cortical neuron lysates were immunoprecipitated with an anti-TBC1D10B antibody and immunoblotted for BAG3. An indicated fraction of cell lysate was used as input control with GAPDH as loading control. Immunoprecipitates were probed for TBC1D10B, BAG3, and Rab35. Vertical dashed lines indicate that intervening lanes were removed. However, all images were from the same blot and exposure. (B) Representative immunostaining of BAG3 (green) and TBC1D10B (red) co-localization in the CA1, counterstained with Hoechst 33342. Scale bars, 20μm. (C-G) Neurons were immunostained for BAG3 (red) and TBC1D10B (green). Overlap of BAG3 with TBC1D10B puncta was observed in the neurites (D) and soma (F) with corresponding line scans (E&G). Arrowheads indicate areas of overlap. Scale bar, 10 μm.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Immunoprecipitation, Control, Immunostaining

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A) BAG3 null HEK293TN cells were transiently transfected with FLAG-TBC1D10B together with empty vector (Control), wild type BAG3 (WT BAG3), or indicated BAG3 mutants. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by blotting for BAG3 and FLAG. Immunoprecipitates were probed for FLAG and BAG3. (B) BAG3 null HEK293TN cells were transiently transfected with V5-HSP70 together with wild type BAG3 (WT) or L462P BAG3 (L462P). Cell lysates were immunoprecipitated with a V5 tag antibody, followed by blotting for BAG3 and V5. (C) HEK293TN cells were transiently transfected with FLAG-TBC1D10B together with an empty vector (Control) or V5-HSP70. Cell lysates were immunoprecipitated with an anti-FLAG antibody, followed by blotting for endogenous BAG3, FLAG, and V5. (D) HEK293TN cells were transient co-expressed with FLAG-TBC1D10B together with an empty vector or V5-HSP70 and treated with 10 μM YM01 or DMSO (vehicle control). Corresponding lysates were collected and immunoprecipitated with anti-FLAG antibody, followed by blotting for BAG3, V5, and FLAG. (E) BAG3 null HEK293TN cells were transiently transfected with V5-HSP70 and FLAG-TBC1D10B. Corresponding lysates were immunoprecipitated with anti-FLAG antibody, followed by blotting for V5 and FLAG. Results of different exposure times for the same blot were separate with a vertical dotted line. Indicated fraction of cell lysate was used as input control. (F) BAG3 null HEK293TN cells were transiently transfected with FLAG-TBC1D10B, V5-HSP70 together with an empty vector (Control) or wildtype BAG3 (BAG3 WT). Cell lysates were immunoprecipitated with anti-FLAG antibody, followed by blotting for BAG3, V5, and FLAG. (G) Cell lysate from F was immunoprecipitation with the 0.5µg IgG or V5 antibody which is saturated by V5-HSP70 and blotted for FLAG. GAPDH was used as a loading control for all input lanes.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transfection, Plasmid Preparation, Control, Immunoprecipitation

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A) Rat cortical neurons were transduced with lentivirus expressing shBAG3 or a scrambled (Scr) version. Cell lysates were immunoprecipitated with an anti-TBC1D10B antibody and immunoblotted for BAG3, TBC1D10B, and Rab35. An 8% fraction of cell lysate was used as input control. (B) Rat cortical neurons were transduced with lentivirus expressing scrambled (Scr) or shBAG3 shRNA. Rab35 activity was examined by incubation of cell lysates with purified GST or GST-RBD35, followed by precipitation with glutathione beads . The precipitated samples were blotted for Rab35 and GST. 4% of the cell lysate was used as input control. (C) BAG3 null HEK293TN cells were transiently transfected with Myc-Rab35 together with empty vector (Control), wild type BAG3 (WT BAG3) or L462P BAG3. Rab35 activity was examined by pull-down with GST-RBD35, as described in B. The precipitated samples were blot for Rab35 and GST. 1.2% of the cell lysate was used as input control. (D) Rat cortical neurons were transduced with lentivirus expressing scrambled (Scr), shBAG3, shTBC1D10B, or both shBAG3 and shTBC1D10B shRNAs. Rab35 activity was examined by pull-down with GST-RBD35. Arrow indicates the GST-RBD35 fusion protein; **, GST-RBD35 degradation products, *, GST protein. GAPDH was used as a loading control for all input lanes.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transduction, Expressing, Immunoprecipitation, Control, shRNA, Activity Assay, Incubation, Purification, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A) Rat cortical neurons were transduced with lentivirus expressing scrambled (Scr) or shTBC1D10B shRNA. Cell lysates were immunoblotted for TBC1D10B, p-Ser262, p-Ser396/404, and p-Thr231 tau. GAPDH is used as a loading control. (B) Relative level of p-tau to total tau. Data are shown as mean ± SEM with unpaired Student’s t-test; *, P<0.05, **, P<0.01. (C-D) Rat cortical neurons were transduced with lentivirus expressing scrambled (Scr) or shBAG3 or both shBAG3 and shTBC1D10B shRNAs. Cell lysates were either immunoblotted for BAG3 and TBC1D10B for validating the knockdown (C) or p-Ser262, p-Ser396/404, and p-Thr231 tau (D). (E) Relative level of p-tau to total tau. Data are shown as mean ± SEM with one-way ANVOA and Tukey’s multiple comparisons test. *, P<0.05, **, P<0.01. For all experiment n=3 for each group. Vertical dotted lines indicate that intervening lanes were removed, however, all images were from the same blot and exposure.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transduction, Expressing, shRNA, Control, Knockdown

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A-F) Rat cortical neurons that were transduced with lentivirus expressing scrambled (Scr) or shBAG3 shRNAs were co-immunostained for Rab35(Green) and Hrs (Red). Overlap of Rab35 with Hrs puncta was observed in the soma (A) and neurites (D). Scale bars, 10 μm. The corresponding line scans are shown at the bottom for A or right for D. Arrowheads indicate areas of overlap. (B, E) Quantification of the co-localization between Rab35 and Hrs based on volume. (C, F) Quantification of co-occurring Rab35 in Hrs using Mander’s coefficient. N=12 neurites from 6 different neurons in each group. Data was presented as mean±SD with unpaired Student’s t-test., *P<0.05, **P<0.01. (G) BAG3 null HEK293TN cells were transfected with BAG3 WT (+BAG3) or empty vector (BAG3 KO), RFP-Hrs, and GFP-Rab35. Live-cell imaging was imaged at 0.1 Hz for 10 minutes. G1 and G2, are representative snapshots of HEKS with or without BAG3. G1’, G1’’ and G2’, G2’’ are 3D kymographs of G1 and G2. The white color indicates the co-localized region of Rab35 and Hrs over time. Scale bars, 20µm. (H) The graph shows the track distance of Hrs puncta in 10 minutes. The distance of the track was binned every 0.1 μm. The percentage of events is defined as the percentage of tracks of Hrs in a certain distance range in one cell. At least 10 cells from each group were analyzed. (p < 0.01, Kolmogorov-Smirnov test). (I) Comparison of co-occurring Rab35 in Hrs using Mander’s co-localization coefficient over time in BAG3 expressing and BAG3 KO cells. Mean±SD, N=12, with unpaired Student’s t-test. **, P<0.01. (J) BAG3 null HEK293TN cells were transfected with Myc-Rab35 and with/without BAG3 and TBC1D10B. Corresponding cell lysates were immunoprecipitated with anti-Myc antibody and immunoblotted for Hrs and Myc. Four percent of the lysates were used for input control. (K-M) BAG3 null HEK293TN cells stably expressing tau were transfected with Myc-Rab35 together with empty vector (Control), wildtype BAG3 (BAG3 WT), or BAG3 mutants. (K) Corresponding lysates were immunoblotted for total tau, Hrs and BAG3 and quantified in (L). N=3 for each group. Data are shown as mean ± SEM with one-way ANOVA and Tukey’s multiple comparisons test. **, P<0.01. n.s. not significant. (M) Cell lysates were immunoprecipitated with Myc antibody and immunoblotted for Hrs. For immunoblots shown in F, G, and I, GAPDH was used as a loading control.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Transduction, Expressing, Transfection, Plasmid Preparation, Live Cell Imaging, Comparison, Immunoprecipitation, Control, Stable Transfection, Western Blot

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: Two-month-old P301S mice were intrahippocampally injected with AAV2/9-eGFP as control or AAV2/9-eGFP-hBAG3 (with a FLAG-Myc N terminal tag) for BAG3 overexpression (BAG3 O/E), and animals were collected at 6 months of age. (A) Images of the AAV injected brains were co-immunostained for BAG3 (red) and Myc tag (purple). The immunofluorescence images showed increased levels of BAG3 in the hippocampal region in the BAG3 O/E brain. Scale bars, 200μm. (B) Hippocampal lysates from control and BAG3 O/E brains were immunoblotted for BAG3. GAPDH is used as a loading control. (C) The graph shows the level of BAG3 normalized to GAPDH and relative to control. N=4 for control, n=5 for BAG3 O/E, Data are shown as mean ± SEM with Student’s t-test, *P<0.05. (D, E) Hippocampal lysates from control and BAG3 O/E brains were blot for total human tau (HT7 and 5A6). The graphs on the right show the quantitative analysis of tau normalized to GAPDH and relative to control. N=4 for control, n=5 for BAG3 O/E. (F) Representative blots of phosphorylated tau (p-Thr231, p-Ser262, and p-Ser396/Ser404) in the hippocampus lysate of control and BAG3 O/E brains. (G) Quantification of the levels of phosphorylated tau in control and BAG3 O/E hippocampal lysates. Data were normalized to the loading control GAPDH and then compared to controls, N=4 for control, n=5 for BAG3 O/E. (H, I) Phosphorylated tau immunofluorescence (p-Ser262 and p-Ser396/Ser404, Green) staining in the CA1 with corresponding intensity quantification relative to controls, N=4. (J) Oligomeric tau (T22 tau, purple) immunofluorescence staining in the CA1 with corresponding quantification, N=3. (K) Pre-tangle and tangle conformation tau (MC1 tau, green) immunofluorescence staining in the CA1 with corresponding quantification relative to controls, N=3. For all images, scale bars, 20μm and data are shown as mean ± SEM with unpaired Student’s t-test;. *, P<0.05; **, P<0.01.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Injection, Control, Over Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: Two-month-old P301S mice were intrahippocampal injected with AAV2/9-GFP as control or AAV2/9-GFP-BAG3-MYC as BAG3 overexpression (BAG3 O/E) and were collected at 6-month old. (a) Representative immunoblots of BAG3 for cerebellum lysate of control and BAG3 O/E brains. (b) The graph shows the quantitative analysis of BAG3 to GAPDH and relative to control. (c, e) Representative blot of total tau (HT5 and 5A6) for cerebellum lysates of control and BAG3 O/E brains. (d, f) The graphs show the quantitative analysis of tau to GAPDH and relative to control. N=4 for control, n=5 for BAG3 O/E. Data are shown as mean ± SEM. Statistical analysis was performed using the student t-test. NS, not significant.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Injection, Control, Over Expression, Western Blot

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: Two-month-old P301S tau mice were intrahippocampally injected with AAV2/9-GFP as control or AAV2/9-GFP-hBAG3 (with a FLAG-Myc N terminal tag) as BAG3 overexpression (BAG3 O/E) and collected at 6-month old. (A) Representative immunofluorescence staining and co-localization of Rab35 (Green) and Hrs (Red) in the CA1. (B) Quantification of co-occurring Rab35 in Hrs using Mander’s co-localization coefficient. (C) Quantification of the co-localization between Rab35 and Hrs based on volume. N=3 with unpaired Student’s t-test.. (D) Representative immunofluorescence staining of p-Ser396/404 tau (green) co-localization with CHMP2B (red) in the CA1. (E) Quantification of co-occurring of CHMP2B in p-Ser396/404 tau using Mander’s coefficient (F) Quantification of the co-localization between p-Ser396/404 tau and CHMP2B. All samples were counterstained with Hoechst 33342 (blue) to visualize the nuclei. Scale bars denote 20 μm. N=4 with unpaired Student’s t-test. *P<0.05, **P<0.01.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: Injection, Control, Over Expression, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: BAG3 regulation of Rab35 mediates the Endosomal Sorting Complexes Required for Transport /endolysosome pathway and tau clearance

doi: 10.1101/2021.01.25.428055

Figure Lengend Snippet: (A) During normal aging, BAG3 level increases, promoting the association of BAG3-HSP70-TBC1D10B. This prevents TBC1D10B from inactivating Rab35, leading to Hrs recruitment and initiation of ESCRT-mediated endosomal tau clearance. (B) In AD brains, however, this increase of BAG3 may be attenuated, which would release TBC1D10B from the BAG3-HSP70-TBC1D10B complex. Free TBC1D10B would thus be able to inactivate Rab35 and prevents endosomal clearance of tau, leading to accumulation over time.

Article Snippet: AAV control virus (AAV9-SYN1-eGFP) and AAV BAG3 overexpression virus (AAV9-SYN1-GFP-T2A-BAG3 [with an N terminal FLAG-Myc tag]) were purchased from Vector Biolabs (Vector Biolabs, PA).

Techniques: