2014 addgene  (Addgene inc)

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: BAG6 interacts with TDP43 proteolytic fragments (A) Proteolytic fragments were expressed in mammalian cells using the ubiquitin reference technique . Co-translational cleavage by cellular deubiquitylases (DUBs) yields N-terminally flag-tagged DHFR-Ub R48 (which contains a K48R mutation to prevent its participation in polyubiquitin chains) and a test fragment (e.g., TDP43 219 ) bearing a specified N-terminal amino acid and a C-terminal FLAG epitope tag. DHFR-Ub R48 serves as an internal reference protein. URT-expressed flag-DHFR-Ub-Val-TDP43 219 -flag was labeled with [ 35 S] Met, followed by denaturing immunoprecipitation with anti-FLAG, SDS-PAGE, and autoradiography. (B) Exogenously expressed TDP43 219 and TDP43 247 were immunoprecipitated (IP) from the detergent-soluble ( S ) and -insoluble ( In ) fractions of HEK293T cells using an anti-FLAG antibody. Endogenous BAG6 was identified in the lysates and IP fractions using an anti-BAG6 antibody. TDP43 219 and TDP43 247 were identified in the IP fractions using an anti-TDP43 antibody. (C) Co-IP of DHFR-Ub, TDP43 219 , and TDP43 247 with wild type BAG6 and BAG6 lacking its N-terminal UBL domain (BAG6ΔUBL) expressed in HEK293T cells. Note that endogenous, full-length TDP43 is not immunoprecipitated by BAG6 or BAG6ΔUBL. Mock , mock-transfected. (D) Relative affinity of TDP43 219 versus TDP43 247 when co-immunoprecipitated using an anti-BAG6 antibody. Experiments were carried out in triplicate. Error bars indicate standard error of the means (SEM). An independent t-test yielded significant differences between TDP43 219 and TDP43 247 , t(4) = -22.9, (∗, p < 0.001). (E) Kyte-Doolittle plot showing regions of hydrophobicity (in green) throughout human full-length TDP43. Hydrophobic regions shaded in light green are exposed in C-terminal fragments of TDP43. NLS , nuclear localization signal, RRM , RNA recognition motif.

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Ubiquitin Proteomics, Mutagenesis, FLAG-tag, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Co-Immunoprecipitation Assay, Transfection

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: BAG6 solubilizes TDP43 219 (A) Upper panel , BAG6 is detected by immunoblot using an anti-BAG6 antibody in soluble lysates of wild type HEK293T cells but not clones that have undergone CRISPR-Cas9-mediated BAG6 ablation (BAG6-KO). Lower panel , coomassie stain of the membrane to indicate relative amounts of lysate loaded onto gel. (B) BAG6-KO cells were transfected with TDP43 219 and increasing amounts of a plasmid encoding wild type BAG6. Cells were lysed and fractionated into detergent-soluble ( Sol ) and -insoluble ( Ins ) fractions. TDP43 219 and endogenous, full-length TDP43 was detected using a C-terminal anti-TDP43 antibody. DHFR-Ub was used as a control. (C) Same as in B, except using BAG6 lacking its N-terminal UBL domain (BAG6ΔUBL). (D) Relative levels of TDP43 219 in soluble fractions of BAG6-KO cells as a result of increasing amounts of BAG6 or BAG6ΔUBL. Experiments were carried out in triplicate. A one-way ANOVA demonstrated significant between group differences in soluble TDP43 219 as a result of increasing concentrations (0, 0.5, 1.0, and 2.0 μg) of BAG6-or BAG6ΔUBL-expressing plasmid (F(6,20) = 14.98, p < 0.01). Fisher LSD post-hoc tests revealed significant differences between samples lacking BAG6 and those containing BAG6 or BAG6ΔUBL. Error bars indicate standard error of the means (SEM). (∗ relative to samples lacking BAG6, p < 0.05).

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Western Blot, Clone Assay, CRISPR, Staining, Membrane, Transfection, Plasmid Preparation, Control, Expressing

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: BAG6 prevents the oligomerization of TDP43 proteolytic fragments HEK293T cells were either mock transfected (−) or transfected with plasmids expressing either TDP43 219 or TDP43 247 in the presence or absence of exogenously overexpressed BAG6. To detect oligomers, cell pellets were treated with 1 mM disuccinimidyl glutarate (DSG) and lysates were fractionated into detergent-soluble and -insoluble (urea-soluble) factions. TDP43 fragments were detected in the soluble and insoluble fractions by immunoblotting using an anti-TDP43 antibody. Endogenous and exogenous BAG6 was detected by immunoblotting using an anti-BAG6 antibody. Note that dimers of TDP43 247 overlap with endogenous nonspecific bands denoted by a sterisks ( lanes 6 and 12 ).

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Transfection, Expressing, Western Blot

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: BAG6 prevents intracellular aggregation of TDP43 219 (A) A modified version of the URT whereby DHFR is replaced by mCherry to identify cells expressing TDP43 219 . (B) Representative images of BAG6-KO cells expressing mCherry-Ub K48R -TDP43 219 in the presence or absence of BAG6. Upper panels, transfected cells are detected by mCherry-Ub red fluorescence. Lower panels, Aggregates were detected using an anti-FLAG antibody and an Alexa fluor488-conjugated secondary antibody. Bars indicate 10μm. DAPI, 4,6-diaminidino-2-phenylindole. (C) Percentage of mCherry positive cells containing detectable aggregates. Error bars indicate standard errors of the means (SEM). Experiments were carried out in triplicate. At least 600 transfected cells were analyzed for each group. A one-way ANOVA revealed a significant effect of BAG6 knockout on protein aggregation (F(3,13) = 13.661, p = 0.001) with Fisher LSD post hoc tests showing significant group differences. Asterisks represent bars that are significantly different from mock-treated BAG6-KO cells (∗, p = 0.025; ∗∗, p = 0.006).

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Modification, Expressing, Transfection, Fluorescence, Knock-Out

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: TDP43 219 interacts BAG6, TRC35, and RNF126 and is associated with RNF126-catalyzed ubiquitylation (A) BAG6-KO cells were transiently transfected with FLAG epitope-tagged TDP43 219 , BAG6, TRC35, UBL4a and RNF126. Upper panels , Proteins interacting with TDP43 219 were detected by IP using anti-TDP43, followed by immunoblot using anti-FLAG. Asterisk , antibody heavy chain. Lower panels , anti-FLAG immunoblot of lysates. (B) BAG6-KO cells were co-transfected with plasmids expressing TDP43 219 , TDP43 247 , and mCherry-Ub (as a control) and either BAG6, RNF126, or BAG6 and RNF126 together. Proteins associated with TDP43 proteolytic fragments were detected by anti-FLAG immunoblot of an anti-TDP43 co-immunoprecipitation. (C) In vitro ubiquitylation reactions containing the indicated components. Total ubiquitylation was detected in reaction mixtures using an anti-ubiquitin antibody. TDP43 219 -specific ubiquitylation was detected by anti-ubiquitin immunoblot of anti-FLAG IP samples. TDP43 219 was detected using an anti-TDP43 antibody. Additional reaction components were identified through coomassie staining of lysate membrane.

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Transfection, FLAG-tag, Western Blot, Expressing, Control, Immunoprecipitation, In Vitro, Ubiquitin Proteomics, Staining, Membrane

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet: BAG6 has a general role in preventing aggregation of neurodegeneration-associated proteolytic fragments (A) FLAG-tagged DHFR-Ub (as a control), TDP43 208 , Tau, βCTF, and 13myc-tagged Aβ were expressed in the presence of overexpressed BAG6 in HEK293T cells. Upper panels , Proteins interacting with BAG6 were detected using an anti-BAG6 co-IP, followed by immunoblot using the indicated antibodies. Lower panels , immunoblot of lysates using the indicated antibodies. Mock , mock-transfected. LC , antibody light chain. (B) IP and lysate fractions from panel A ( lanes 7 through 12 ) immunoblotted with an anti-TDP43 antibody. Asterisk , endogenous, cross-reacting band. (C and D) BAG6-KO cells expressing either TDP43 208 or βCTF in the presence or absence of BAG6. Cells were lysed and fractionated into detergent-soluble ( Sol ) and -insoluble ( Ins ) fractions. BAG6, TDP43 208 and βCTF were detected using an anti-FLAG antibody. Anti-β-actin and anti-fibrillarin was used as loading controls for the soluble and insoluble fractions, respectively (D) Model of BAG6 role in preventing the aggregation of proteolytic fragments. Limited proteolysis ( indicated by scissors ) of various cellular proteins generates misfolded proteolytic fragments with solvent-exposed hydrophobic regions ( red portion of protein ). In the absence of their degradation, these fragments self-associate to form oligomers and insoluble aggregates that are associated with neurodegeneration. Alternatively, hydrophobicity is bound by BAG6 which prevents fragment oligomerization and aggregation. The BAG6 complex can recruit various E3 Ub-ligases—e.g., RNF126—to facilitate client ubiqiutylation and proteasome-mediated degradation.

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Control, Co-Immunoprecipitation Assay, Western Blot, Transfection, Expressing, Solvent

Journal: iScience

Article Title: BAG6 prevents the aggregation of neurodegeneration-associated fragments of TDP43

doi: 10.1016/j.isci.2022.104273

Figure Lengend Snippet:

Article Snippet: pRK5-FLAG-Bag6: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6 under the control of CMV promoter , , Addgene Cat#61836.

Techniques: Magnetic Beads, Virus, Recombinant, Expressing, Plasmid Preparation, Cloning, Control

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 1. (A) Immunoblot analysis of endogenous G0S2 protein half-life in neonatal rat cardiomyocytes. Cells were treated with cycloheximide (CHX) (100 µg/ml) at the indicated times. (B) Immunoblotting of cardiomyocytes treated with proteasome inhibitors (MG132: 10 µM; Lactacystin: 10 µM; PS-341: 0.3 µM) or lysosomal inhibitors (chloroquine: 100 µM; NH4Cl: 10 mM). Four hours after the treatment, cells were harvested and subjected to immunoblot analysis. (C) Schematic workflow of siRNA library screening. C2C12 cells stably expressing EGFP-fused G0S2 (C2C12/EGFP-G0S2 cells) or EGFP-fused CL1 degron peptide (C2C12/EGFP-CL1 cells) were transfected with the indicated siRNA. Seventy-two hours after transfection, cells were imaged and fluorescence intensities of EGFP were analyzed by IN Cell Analyzer 6000. See also Fig. S1. (D) Distribution of siRNAs ranked according to values of log2 fold change (FC) of EGFP-G0S2 intensity that is averaged and normalized to the siCTL in three independent experiments. Black and red dots represent negative and positive hits, respectively. (E) Scatter plot of the log2FC of EGFP intensity in C2C12/EGFP-CL1 cells and the number of C2C12/EGFP-G0S2 cells, both averaged and normalized to the siCTL in three independent experiments. Each dot represents one siRNA pool targeting one gene. (F) C2C12 cell lines stably expressing HA-tagged G0S2 (C2C12/HA-G0S2 cells) were transfected with 30 nM of either siCTL or siRNF126, as indicated. After 48 h incubation, total RNA was extracted and analyzed by qPCR. Data represent mean values ± S.D. (n=3). ***P<0.001. (G) Seventy-two hours after transfection of siRNA as in (F), cells were harvested and subjected to immunoblot analysis. RNF126 was detected by a RNF126 #A antibody.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Western Blot, Library Screening, Stable Transfection, Expressing, Transfection, Fluorescence, Incubation

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 4. (A–E) The indicated siRNA was expressed for 72 h in (A) cardiomyocytes or (B–E) C2C12/HA-G0S2 cells. Cells were harvested and subjected to immunoblotting. (C) Cells were solubilized and ubiquitinated proteins were enriched by TUBE pull-down assay. (D) Immunoblotting of cells that were treated with cycloheximide at the indicated times (0–120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0min) and is shown graphically. The asterisks denote statistical significance comparing siCTL- and siBAG6-treated cells. Data represent mean values ± S.D. (n=3). *P<0.05. **P<0.01. (F) Representative YFP/CFP ratiometric pseudocolored images of Mit-ATeam fluorescence in cardiomyocytes expressing the indicated siRNA and adenoviral Mit-ATeam for 48 h. (Scale bar, 10 µm) (G) YFP/CFP emission ratio plots of Mit-ATeam fluorescence in cardiomyocytes transfected siCTL (n=18), siBAG6 #1 (n=20) or siBAG6 #3 (n=20) during hypoxia. All of measurements were normalized to the ratio at time 0 and compared between cardiomyocytes with siCTL, siBAG6 #1 and #3 at each time point. Data represent mean values ± SEM. n.s., not significant.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Western Blot, Pull Down Assay, Fluorescence, Expressing, Transfection

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 5. (A) Schematic diagram of generated mutants. (B) C2C12 cells were transfected with the indicated G0S2 mutants. After 4 h treatment with DMSO (D : 0.1%) or MG132 (MG : 10 µM), cells were harvested and subjected to immunoblot analysis. (C) C2C12/HA-G0S2 WT or E44A cells were solubilized and ubiquitinated proteins were enriched by TUBE2 pull-down assay. (D) C2C12/HA-G0S2 WT or E44A cells were treated with cycloheximide at the indicated times (0-120 min). (E) Each protein level in (D) was densitometrically quantified (normalized to 0 min) and is shown graphically. The asterisks denote statistical significance comparing C2C12/HA-G0S2 WT and E44A cells. Data represent mean values ± S.D. (n=3). **P<0.01. (F) Immunoprecipitation of HA-G0S2 in HEK293T cells. Cells expressing HA-tagged G0S2 WT or E44A were harvested and immunoprecipitated with anti-HA antibody. (G) C2C12 cells expressing HA-G0S2 WT or E44A were transfected with the indicated siRNA for 48 h. After solubilizing with the buffer containing 1% Triton X-100, lysates were centrifuged and the supernatant (S) and pellet (P) fractions were analyzed by immunoblotting. The pellet contains the detergent-insoluble protein aggregates. GAPDH and vimentin are used as the detergent-soluble and -insoluble fractions, respectively.

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: Generated, Transfection, Western Blot, Pull Down Assay, Immunoprecipitation, Expressing

Journal: Journal of Biological Chemistry

Article Title: A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

doi: 10.1074/jbc.ra119.008544

Figure Lengend Snippet: Figure 6. (A) In vitro ubiquitination assay in the presence or absence of purified recombinant UBE1 (E1), UBCH5B (E2), GST-RNF126 (E3) and BAG6, together with His-tagged ubiquitin, ATP and HA-G0S2 as a substrate. The mixture was incubated for 60 min at 37oC, and the reaction was stopped by the addition of 3x sample buffer. See also Fig. S4. (B) In vitro ubiquitination assay as in (A) except for the use of GST-RNF126 WT or C231A/C234A (CA) mutant, and HA-G0S2 WT, E44A, or lysine-less mutant (6KR).

Article Snippet: The plasmid encoding human BAG6 (pRK5-FLAG-BAG6) was obtained from Addgene (#61836). siRNA library screening C2C12 cell lines stably expressing EGFP fused G0S2 or CL1 degron for siRNA library screening were generated as follows: lentiviral particles encoding EGFP-G0S2 or EGFP-CL1 (ACKNWFSSLSHFVIHL) were generated as described above, and then infected into C2C12 cells, followed by puromycin selection (5 μg/ml: SigmaAldrich).

Techniques: In Vitro, Ubiquitin Proteomics, Purification, Recombinant, Incubation, Mutagenesis