filamentous phage rscq based r pseudosolanacearum cloning vector prscq1  (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-Bag6ΔUBL: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6ΔUBL under the control of CMV promoter , , Addgene Cat#61837.

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-Bag6ΔUBL: Amp R ; Neo R ; pRK5-based plasmid encoding f BAG6ΔUBL under the control of CMV promoter , , Addgene Cat#61837.

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: 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: GST-RNF126: Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 , , Addgene Cat#138643.

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: GST-RNF126: Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 , , Addgene Cat#138643.

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: GST-RNF126: Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 , , Addgene Cat#138643.

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

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: 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: pAAV-Ef1a-Cre: Amp R ; AAV-based plasmid encoding Cre recombinase under the control of EF1α promoter , , Addgene Cat#55636.

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

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: GST-RNF126 C231A : Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 C231A , , Addgene Cat#138644.

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: GST-RNF126 C231A : Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 C231A , , Addgene Cat#138644.

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: GST-RNF126 C231A : Amp R ; pGEX-4T3 based plasmid for bacterial expression of GST RNF126 C231A , , Addgene Cat#138644.

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

Journal:

Article Title: Genomic and Genetic Analysis of Bordetella Bacteriophages Encoding Reverse Transcriptase-Mediated Tropism-Switching Cassettes

doi: 10.1128/JB.186.5.1503-1517.2004

Figure Lengend Snippet: Bacterial strains and plasmids a

Article Snippet: When appropriate, media were supplemented with 40 μg of 5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside (X-Gal) per ml. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristics Source or reference B. bronchiseptica RB50 Isolated in 1992 at UCLA from a rabbit, Sm r 2, 9 RB30 Isolated in 1992 at UCLA from a rabbit, Sm r J.F.M. lab collection RB50Gm RB50 with Gm r cassette This work BB3464 Isolated from feline respiratory tract in 1988 at UC Davis J.F.M. lab collection ML6401 BPP-1 lysogen of RB50 This work ML6403 BIP-1 lysogen of RB50 This work ML6405 BMP-1 lysogen of RB50 This work RB53 RB50 bvgS -C3 2, 9 RB54 RB50 ΔbvgAS 2, 9 ML83DMIN ML6401 BPP-1Δ bpm This work ML83DM3AN ML6403 BIP-1Δ bpm This work ML83DM5A ML6405 BMP-1Δ bpm This work ML89-D36-1A ML6401 BPP-1Δ bbp36 This work ML89-D36-3A ML6403 BIP-1Δ bbp36 This work ML89-D36-5A ML6405 BMP-1Δ bbp36 This work MH406 RB50Rf wbmD671 ::mTn 5 - lacZ1 This work G2-15 RB50Rf fhaB3125 ::mTn 5-lacZ1 This work E. coli DH5α F − hsdR17 supE44 thi-1 recA1 gyrA relA1 Δ( argF-lac ) U169 φ80d lacZ Δ M15 Gibco-BRL XL1 recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lac [F′ proAB + lacI q ZΔM15::Tn 10 ] Stratagene DH5αλpir DH5α λ pir J.F.M. lab collection Plasmids pMLG13 oriT Amp r Gm r ColE1 MCS from pBluescript II KS+ This work pML68-G3 1.4-kb Eco RI- Bam HI insert containing the complete c I repressor CDS cloned into pMLG13 This work pRK2013 Km r ori ColE1 RK2-Mob + RK2-Tra + 11 pML89-D36-6 pRE112-based Δ orf36 allelic exchange vector This work pML83-112M3A pRE112-based Δ bpm allelic exchange vector This work pBBR1MCS Cm r broad-host-range plasmid ATCC pML93-bpm106 pBBR1MCS-based plasmid expressing bpm This work pRE112 Cm r allelic exchange vector ATCC Open in a separate window a UCLA, University of California, Los Angeles; UC, University of California; MCS, multiple cloning site; CDS, coding sequence; ATCC, American Type Culture Collection.

Techniques: Plasmid Preparation, Isolation, Clone Assay, Expressing