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Addgene inc pcdna3 1 multiple cloning site
Pcdna3 1 Multiple Cloning Site, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna3+1+multiple+cloning+site/pmc10785701-393-19-15?v=Addgene+inc
Average 93 stars, based on 15 article reviews

pcdna3 1 multiple cloning site - by Bioz Stars, 2026-07

93/100 stars

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OGA-mBirA fusion proteins express, maintain catalytic activity, and biotinylate proteins in vivo. A, schematic of the OGA-mBirA fusion proteins. CD and AD represent the catalytic (β-N-acetylglucosaminidase) domain and acetyltransferase-like domain of OGA, respectively. B–D, U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with or without biotin (25 μm, 16 h) or TMG (100 nm, 20 h) as indicated. Proteins were extracted in TCL buffer. B, equal amounts of protein (10 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: OGA, HA, Myc, and actin. n = 3. C, desalted lysates were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 3, representative data from one experiment is shown. Error bars indicate the intra-assay standard deviation from two technical replicates. D, equal amounts of protein (4.5 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 2. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA fusion proteins express, maintain catalytic activity, and biotinylate proteins in vivo. A, schematic of the OGA-mBirA fusion proteins. CD and AD represent the catalytic (β-N-acetylglucosaminidase) domain and acetyltransferase-like domain of OGA, respectively. B–D, U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with or without biotin (25 μm, 16 h) or TMG (100 nm, 20 h) as indicated. Proteins were extracted in TCL buffer. B, equal amounts of protein (10 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: OGA, HA, Myc, and actin. n = 3. C, desalted lysates were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 3, representative data from one experiment is shown. Error bars indicate the intra-assay standard deviation from two technical replicates. D, equal amounts of protein (4.5 μg) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 2. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Article Snippet: The following DNA constructs were gifts (Kyle Roux, Sanford Research ( 39 )): pcDNA3.1 Myc-mBirA multiple cloning site (MCS; plasmid 35700; Addgene, Cambridge, MA) and pcDNA3.1 MCS-mBirA-HA (plasmid 36047; Addgene).

Techniques: Activity Assay, In Vivo, Transfection, SDS Page, Western Blot, Intra Assay, Standard Deviation, Migration

OGA-mBirA fusion proteins localize to and biotinylate proteins in the nucleus, cytoplasm, and mitochondria of U2OS cells. U2OS cells were transfected with pcDNA3.1 (A), OGA-mBirA-HA (B–D), or Myc-mBirA-OGA (E–G), treated with (A, C, D, F, and G) or without (B and E) biotin (25 μm, 16 h), and treated with (D and G) or without (A–C, E, and F) H2O2 (2.5 mm, 2 h). Cells were fixed, permeabilized, and stained for BirA and biotin. Nuclei and mitochondria were stained with Hoechst 33342 and MitoTracker Orange CMTMRos, respectively. White triangles indicate co-localization (orange) of MitoTracker and OGA (B and E). Images were acquired at ×63 magnification on a Zeiss Axio Examiner 710NLO-Meta multiphoton microscope. n = 3. Scale bar, 15 μm.

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA fusion proteins localize to and biotinylate proteins in the nucleus, cytoplasm, and mitochondria of U2OS cells. U2OS cells were transfected with pcDNA3.1 (A), OGA-mBirA-HA (B–D), or Myc-mBirA-OGA (E–G), treated with (A, C, D, F, and G) or without (B and E) biotin (25 μm, 16 h), and treated with (D and G) or without (A–C, E, and F) H2O2 (2.5 mm, 2 h). Cells were fixed, permeabilized, and stained for BirA and biotin. Nuclei and mitochondria were stained with Hoechst 33342 and MitoTracker Orange CMTMRos, respectively. White triangles indicate co-localization (orange) of MitoTracker and OGA (B and E). Images were acquired at ×63 magnification on a Zeiss Axio Examiner 710NLO-Meta multiphoton microscope. n = 3. Scale bar, 15 μm.

Techniques: Transfection, Staining, Microscopy

OGA-mBirA-HA and Myc-mBirA-OGA biotinylate proximal proteins differentially in response to oxidative stress. U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with biotin (25 μm, 16 h) in the presence or absence of H2O2 (2.5 mm, 2 h). A, equal amounts of protein (5 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 4. B, densitometric total lane profiles for each lane from the biotin signal in A. Asterisks are used to highlight a subset of the biotinylated signals that are altered by oxidative stress. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA-mBirA-HA and Myc-mBirA-OGA biotinylate proximal proteins differentially in response to oxidative stress. U2OS cells were transfected with pcDNA3.1, OGA-mBirA-HA, or Myc-mBirA-OGA and treated with biotin (25 μm, 16 h) in the presence or absence of H2O2 (2.5 mm, 2 h). A, equal amounts of protein (5 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, O-GlcNAc, OGA, HA, Myc, and actin. n = 4. B, densitometric total lane profiles for each lane from the biotin signal in A. Asterisks are used to highlight a subset of the biotinylated signals that are altered by oxidative stress. Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Techniques: Transfection, Lysis, SDS Page, Western Blot, Migration

$SILAC-BioID-MS/MS strategy used to identify the basal and oxidative stress-dependent interactome of OGA. A, U2OS cells were labeled with light, medium, or heavy isotopes of arginine and lysine for six generations. In experiment 1, cells were transfected with pcDNA3.1 (heavy) or OGA-mBirA-HA (light, medium), treated with biotin (25 μm, 16 h), and treated with vehicle (medium, heavy) or H2O2 (light; 2.5 mm, 2 h, n = 1). In experiment 2, Myc-mBirA-OGA was transfected in replacement of OGA-mBirA-HA. For each experiment, proteins were extracted in denaturing TCL buffer and combined in equal amounts. The biotinylated proteins were isolated on NeutrAvidin-agarose in denaturing conditions, eluted in 2% (w/v) SDS (95 °C), and precipitated with acetone. Peptides were generated by trypsin and LysC digestion, separated by basic reversed phase (bRP) fractionation, and identified by mass spectrometry (LC-ESI-MS/MS). Subsequently, protein-protein interactions were validated by co- IP and Western blotting. B, for the SILAC experiments (n = 1, each), equal amounts of protein (10 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, OGA, HA, Myc, and actin. Protein load was assessed by total protein stain (Sypro Ruby) and by Western blotting (actin). Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.$

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: SILAC-BioID-MS/MS strategy used to identify the basal and oxidative stress-dependent interactome of OGA. A, U2OS cells were labeled with light, medium, or heavy isotopes of arginine and lysine for six generations. In experiment 1, cells were transfected with pcDNA3.1 (heavy) or OGA-mBirA-HA (light, medium), treated with biotin (25 μm, 16 h), and treated with vehicle (medium, heavy) or H2O2 (light; 2.5 mm, 2 h, n = 1). In experiment 2, Myc-mBirA-OGA was transfected in replacement of OGA-mBirA-HA. For each experiment, proteins were extracted in denaturing TCL buffer and combined in equal amounts. The biotinylated proteins were isolated on NeutrAvidin-agarose in denaturing conditions, eluted in 2% (w/v) SDS (95 °C), and precipitated with acetone. Peptides were generated by trypsin and LysC digestion, separated by basic reversed phase (bRP) fractionation, and identified by mass spectrometry (LC-ESI-MS/MS). Subsequently, protein-protein interactions were validated by co- IP and Western blotting. B, for the SILAC experiments (n = 1, each), equal amounts of protein (10 μg; denaturing TCL lysis) were separated by SDS-PAGE, and the following were detected by Western blotting: biotin, OGA, HA, Myc, and actin. Protein load was assessed by total protein stain (Sypro Ruby) and by Western blotting (actin). Migration of endogenous OGA (e), mBirA-tagged OGA (b), and the molecular mass (MW) markers are indicated.

Techniques: Tandem Mass Spectroscopy, Labeling, Transfection, Isolation, Generated, Fractionation, Mass Spectrometry, Co-Immunoprecipitation Assay, Western Blot, Lysis, SDS Page, Staining, Migration

$Oxidative stress induces the association of OGA with FAS, FLNA, HSC70, and OGT. U2OS cells were treated with Vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 3. A, anti-OGA antibody (IP: OGA; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous OGA from NETN cell lysates (500 μg), of which 1.5–2% (input) and 30–40% (immunoprecipitate) were analyzed by SDS-PAGE. OGA, FAS, FLNA, HSC70, OGT (positive control), and actin (loading/negative control) were detected by Western blotting. B, anti-FAS antibody (IP: FAS; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous FAS from NETN cell lysates (250 μg), of which 3% (input) and 60% (immunoprecipitate) were analyzed by SDS-PAGE. FAS, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. C, U2OS cells were transfected with pcDNA3.1 (control) or pCMV-SPORT6 V5-FAS (test). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (300 μg), of which 1.7% (input) and 33.3% (immunoprecipitate) were analyzed by SDS-PAGE. V5, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. A–C, FAS (CST) and FAS (NB) represent anti-FAS antibody from Cell Signaling Technology and Novus Biologicals, respectively. To ensure that images were in the linear range, Western blot exposures from the input and immunoprecipitated fractions are often different. The exposure lengths for the test and control isotype antibody immunoprecipitates are always identical. The migration of molecular mass (MW) markers is indicated.$

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: Oxidative stress induces the association of OGA with FAS, FLNA, HSC70, and OGT. U2OS cells were treated with Vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 3. A, anti-OGA antibody (IP: OGA; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous OGA from NETN cell lysates (500 μg), of which 1.5–2% (input) and 30–40% (immunoprecipitate) were analyzed by SDS-PAGE. OGA, FAS, FLNA, HSC70, OGT (positive control), and actin (loading/negative control) were detected by Western blotting. B, anti-FAS antibody (IP: FAS; top panel) or a rabbit isotype control immunoglobulin (IP: IgG; middle panel) was used to enrich endogenous FAS from NETN cell lysates (250 μg), of which 3% (input) and 60% (immunoprecipitate) were analyzed by SDS-PAGE. FAS, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. C, U2OS cells were transfected with pcDNA3.1 (control) or pCMV-SPORT6 V5-FAS (test). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (300 μg), of which 1.7% (input) and 33.3% (immunoprecipitate) were analyzed by SDS-PAGE. V5, OGA, HSC70, OGT, and actin (loading/negative control) were detected by Western blotting. A–C, FAS (CST) and FAS (NB) represent anti-FAS antibody from Cell Signaling Technology and Novus Biologicals, respectively. To ensure that images were in the linear range, Western blot exposures from the input and immunoprecipitated fractions are often different. The exposure lengths for the test and control isotype antibody immunoprecipitates are always identical. The migration of molecular mass (MW) markers is indicated.

Techniques: SDS Page, Positive Control, Negative Control, Western Blot, Transfection, Immunoprecipitation, Migration

$OGA exhibits reduced catalytic activity when bound to FAS. U2OS cells stably overexpressing pcDNA3.1 (control) or pcDNA3.1 V5-FAS (test) were treated with vehicle (V) or H2O2 (2.5 mm, 2 h). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (1.6 mg). n = 3. A and B, V5, OGA, and actin (loading/negative control) were detected by Western blotting. Western blots for each antibody were exposed for equal lengths of time. A, analysis of the inputs and unbound fractions. B, analysis of the bound fractions (31.25%). C, OGA activity was measured in the inputs and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min and then normalized to the pcDNA3.1 vehicle-treated sample. D, OGA activity was assessed in the input, bound (on-bead, 31.25%), and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min/μg using densitometric analysis of OGA in the input serial dilutions and the bound fractions, and then normalized to the pcDNA3.1 vehicle-treated sample. Only the data from FAS-overexpressing cells treated with H2O2 are shown, as OGA protein and activity was absent in the bound fraction of the other samples. Data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Tukey's MCT, and differences were considered statistically significant at p ≤ 0.0001 (****). The migration of molecular mass (MW) markers is indicated.$

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: OGA exhibits reduced catalytic activity when bound to FAS. U2OS cells stably overexpressing pcDNA3.1 (control) or pcDNA3.1 V5-FAS (test) were treated with vehicle (V) or H2O2 (2.5 mm, 2 h). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (1.6 mg). n = 3. A and B, V5, OGA, and actin (loading/negative control) were detected by Western blotting. Western blots for each antibody were exposed for equal lengths of time. A, analysis of the inputs and unbound fractions. B, analysis of the bound fractions (31.25%). C, OGA activity was measured in the inputs and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min and then normalized to the pcDNA3.1 vehicle-treated sample. D, OGA activity was assessed in the input, bound (on-bead, 31.25%), and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min/μg using densitometric analysis of OGA in the input serial dilutions and the bound fractions, and then normalized to the pcDNA3.1 vehicle-treated sample. Only the data from FAS-overexpressing cells treated with H2O2 are shown, as OGA protein and activity was absent in the bound fraction of the other samples. Data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Tukey's MCT, and differences were considered statistically significant at p ≤ 0.0001 (****). The migration of molecular mass (MW) markers is indicated.

Techniques: Activity Assay, Stable Transfection, Negative Control, Western Blot, Fluorescence, Migration

FAS overexpression increases O-GlcNAcylation during oxidative stress. U2OS cells stably overexpressing pcDNA3.1 or pcDNA3.1 V5-FAS were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 5. A, NETN lysates (∼7.5 μg) were analyzed by SDS-PAGE. O-GlcNAc, OGA, OGT, V5, and actin were detected by Western blotting. Total protein stain (colloidal Coomassie G-250) was used to assess protein load. The migration of molecular mass (MW) markers is indicated. B, quantitation of O-GlcNAc levels normalized to G-250. C, quantitation of OGA expression normalized to G-250. D, quantitation of OGT expression normalized to G-250. B–D, data are presented as the mean ± S.E. Significance was determined by RM-2ANOVA followed by Sidak's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Journal: The Journal of Biological Chemistry

Article Title: Fatty acid synthase inhibits the O- GlcNAcase during oxidative stress

doi: 10.1074/jbc.M116.760785

Figure Lengend Snippet: FAS overexpression increases O-GlcNAcylation during oxidative stress. U2OS cells stably overexpressing pcDNA3.1 or pcDNA3.1 V5-FAS were treated with vehicle (V) or H2O2 (2.5 mm, 1–3 h). n = 5. A, NETN lysates (∼7.5 μg) were analyzed by SDS-PAGE. O-GlcNAc, OGA, OGT, V5, and actin were detected by Western blotting. Total protein stain (colloidal Coomassie G-250) was used to assess protein load. The migration of molecular mass (MW) markers is indicated. B, quantitation of O-GlcNAc levels normalized to G-250. C, quantitation of OGA expression normalized to G-250. D, quantitation of OGT expression normalized to G-250. B–D, data are presented as the mean ± S.E. Significance was determined by RM-2ANOVA followed by Sidak's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***).

Techniques: Over Expression, Stable Transfection, SDS Page, Western Blot, Staining, Migration, Quantitation Assay, Expressing

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