Structured Review

TaKaRa egfp sequence
Localization of <t>FSP27-eGFP</t> to lipid droplets in 293T cells and 3T3-L1 adipocytes. Fluorescent images of 293T cells transfected with <t>pEGFP-N1</t> ( A ) or pEGFP-FSP27 ( B ). Nuclei labeled with DAPI ( left panel ), neutral lipid stained with Nile Red ( 2nd panel
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Images

1) Product Images from "Fat-specific Protein 27 Regulates Storage of Triacylglycerol *Fat-specific Protein 27 Regulates Storage of Triacylglycerol * S⃞"

Article Title: Fat-specific Protein 27 Regulates Storage of Triacylglycerol *Fat-specific Protein 27 Regulates Storage of Triacylglycerol * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M708323200

Localization of FSP27-eGFP to lipid droplets in 293T cells and 3T3-L1 adipocytes. Fluorescent images of 293T cells transfected with pEGFP-N1 ( A ) or pEGFP-FSP27 ( B ). Nuclei labeled with DAPI ( left panel ), neutral lipid stained with Nile Red ( 2nd panel
Figure Legend Snippet: Localization of FSP27-eGFP to lipid droplets in 293T cells and 3T3-L1 adipocytes. Fluorescent images of 293T cells transfected with pEGFP-N1 ( A ) or pEGFP-FSP27 ( B ). Nuclei labeled with DAPI ( left panel ), neutral lipid stained with Nile Red ( 2nd panel

Techniques Used: Transfection, Labeling, Staining

2) Product Images from "Gene therapy targeting SARM1 blocks pathological axon degeneration in mice"

Article Title: Gene therapy targeting SARM1 blocks pathological axon degeneration in mice

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20181040

SARM1-CDN protects from AxD in vivo with efficacy similar to SARM1-KO. (A and B) Representative photomicrographs of toluidine blue–stained semithin cross sections of the right sural nerve 5 d after transection of the sciatic nerve in mice injected with vector (A; n = 4) or SARM1-CDN (B; n = 5). A′ and B′ show enlargements of areas indicated by rectangles in A and B. The arrow and arrowhead indicate lipid-laden histiocytes and myelin debris, respectively. (C and D) Representative electron micrographs of the right sural nerve of a mouse injected with EGFP vector showing complete loss of internal nerve architecture (C) whereas unmyelinated (asterisks) and myelinated axons are preserved after SARM1-CDN injection (D). C′ and D′ show enlargements of areas indicated by rectangles in C and D. (E and F) All axons in cross sections of the entire sural nerve were counted in wild-type mice injected with vector ( n = 4 in E; n = 3 in F) or SARM1-CDN ( n = 5 in E, n = 3 in F) or in SARM1-KO mice ( n = 5 in E and F) and expressed as percentage of axon numbers of the respective intact contralateral sides at 5 (E) and 10 (F) d after transection. Data are presented as mean ± SE (SEM), tested with a one-way ANOVA, which shows significant main effects in E (F(2,11) = 97.13, P
Figure Legend Snippet: SARM1-CDN protects from AxD in vivo with efficacy similar to SARM1-KO. (A and B) Representative photomicrographs of toluidine blue–stained semithin cross sections of the right sural nerve 5 d after transection of the sciatic nerve in mice injected with vector (A; n = 4) or SARM1-CDN (B; n = 5). A′ and B′ show enlargements of areas indicated by rectangles in A and B. The arrow and arrowhead indicate lipid-laden histiocytes and myelin debris, respectively. (C and D) Representative electron micrographs of the right sural nerve of a mouse injected with EGFP vector showing complete loss of internal nerve architecture (C) whereas unmyelinated (asterisks) and myelinated axons are preserved after SARM1-CDN injection (D). C′ and D′ show enlargements of areas indicated by rectangles in C and D. (E and F) All axons in cross sections of the entire sural nerve were counted in wild-type mice injected with vector ( n = 4 in E; n = 3 in F) or SARM1-CDN ( n = 5 in E, n = 3 in F) or in SARM1-KO mice ( n = 5 in E and F) and expressed as percentage of axon numbers of the respective intact contralateral sides at 5 (E) and 10 (F) d after transection. Data are presented as mean ± SE (SEM), tested with a one-way ANOVA, which shows significant main effects in E (F(2,11) = 97.13, P

Techniques Used: In Vivo, Staining, Mouse Assay, Injection, Plasmid Preparation

SARM1-CDN efficiently transduces DRGs in vivo and protects from AxD. (A) Top: Schematic of the AAV vector expressing human SARM1-CDN under control of the neuron-specific human synapsin promoter (Syn-SARM1-CDN-EGFP). Bottom: Schematic of the EGFP vector (Syn-EGFP) used for control experiments. WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; ITR, inverted terminal repeats. (B) AAV8-Syn-SARM1-CDN-EGFP or EGFP vector (AAV8-Syn-EGFP) were injected intrathecally (i.t.) into mice at postnatal day 11 or 12 (P11/12). 5 wk later, the right sciatic nerve was transected, and 5 d later, tissue was collected for analysis. (C) Representative micrographs taken in situ of (from left to right) DRGs (asterisk) attached to the spinal cord (SC), the left (uninjured) sciatic nerve (arrow) with its branches (arrowheads), and intercostal nerves (white arrowhead) expressing GFP 5.5 wk after injection with AAV8-Syn-SARM1-CDN-EGFP; m, muscle. Bars, 2 mm. (D) Representative confocal image of a 6-µm-thick section of a DRG after injecting EGFP vector (left column; Syn-EGFP) or SARM1-CDN (right column; Syn-SARM1-DN-EGFP). Sections were stained with PGP9.5 (red; DRG neurons) and anti-GFP (green; construct expression) and coverslipped with Vectamount containing DAPI (blue; nuclear marker). (E) Representative confocal image of a 6-µm-thick section of the right (transected) sciatic nerve taken 5 d after cut in mice injected with the EGFP vector (left column; Syn-EGFP) or SARM1-CDN (right column; Syn-SARM1-CDN-EGFP). Sections were stained with antibodies to Neurofilament 200 (NF) and peripherin (red; axonal markers) and green fluorescent protein (green; construct expression) and mounted with Vectashield containing DAPI (blue; nuclear marker). Bars, 50 µm (D and E).
Figure Legend Snippet: SARM1-CDN efficiently transduces DRGs in vivo and protects from AxD. (A) Top: Schematic of the AAV vector expressing human SARM1-CDN under control of the neuron-specific human synapsin promoter (Syn-SARM1-CDN-EGFP). Bottom: Schematic of the EGFP vector (Syn-EGFP) used for control experiments. WPRE, woodchuck hepatitis virus posttranscriptional regulatory element; ITR, inverted terminal repeats. (B) AAV8-Syn-SARM1-CDN-EGFP or EGFP vector (AAV8-Syn-EGFP) were injected intrathecally (i.t.) into mice at postnatal day 11 or 12 (P11/12). 5 wk later, the right sciatic nerve was transected, and 5 d later, tissue was collected for analysis. (C) Representative micrographs taken in situ of (from left to right) DRGs (asterisk) attached to the spinal cord (SC), the left (uninjured) sciatic nerve (arrow) with its branches (arrowheads), and intercostal nerves (white arrowhead) expressing GFP 5.5 wk after injection with AAV8-Syn-SARM1-CDN-EGFP; m, muscle. Bars, 2 mm. (D) Representative confocal image of a 6-µm-thick section of a DRG after injecting EGFP vector (left column; Syn-EGFP) or SARM1-CDN (right column; Syn-SARM1-DN-EGFP). Sections were stained with PGP9.5 (red; DRG neurons) and anti-GFP (green; construct expression) and coverslipped with Vectamount containing DAPI (blue; nuclear marker). (E) Representative confocal image of a 6-µm-thick section of the right (transected) sciatic nerve taken 5 d after cut in mice injected with the EGFP vector (left column; Syn-EGFP) or SARM1-CDN (right column; Syn-SARM1-CDN-EGFP). Sections were stained with antibodies to Neurofilament 200 (NF) and peripherin (red; axonal markers) and green fluorescent protein (green; construct expression) and mounted with Vectashield containing DAPI (blue; nuclear marker). Bars, 50 µm (D and E).

Techniques Used: In Vivo, Plasmid Preparation, Expressing, Injection, Mouse Assay, In Situ, Staining, Construct, Marker

Identification of SARM1 dominant-negative transgenes. (A) Schematic representation of the domain structure of human SARM1. Individual point mutations are indicated by red triangles. Dotted segments indicate deleted regions. deltaTIR, aa 1–27 and 560–724 deleted; mt, mitochondrial binding sequence; ARM, HEAT/Armadillo motif; SAM, sterile α motif. (B) Axons of wild-type DRG neurons expressing the indicated constructs or axons of SARM1-KO DRG neurons expressing EGFP vector were transected and imaged using high-throughput automated imaging at indicated time points. AxD was quantified using a DI, which ranges from 0 (perfectly intact) to 1 (perfectly fragmented). Shown are means ± SE (SEM) of three independent experiments. Data were tested with a two-way ANOVA showing significant main effects of groups F(6,14) = 25.57; P
Figure Legend Snippet: Identification of SARM1 dominant-negative transgenes. (A) Schematic representation of the domain structure of human SARM1. Individual point mutations are indicated by red triangles. Dotted segments indicate deleted regions. deltaTIR, aa 1–27 and 560–724 deleted; mt, mitochondrial binding sequence; ARM, HEAT/Armadillo motif; SAM, sterile α motif. (B) Axons of wild-type DRG neurons expressing the indicated constructs or axons of SARM1-KO DRG neurons expressing EGFP vector were transected and imaged using high-throughput automated imaging at indicated time points. AxD was quantified using a DI, which ranges from 0 (perfectly intact) to 1 (perfectly fragmented). Shown are means ± SE (SEM) of three independent experiments. Data were tested with a two-way ANOVA showing significant main effects of groups F(6,14) = 25.57; P

Techniques Used: Dominant Negative Mutation, Binding Assay, Sequencing, Expressing, Construct, Plasmid Preparation, High Throughput Screening Assay, Imaging

SARM1-CDN potently inhibits wild-type SARM1 function. (A) Degeneration of wild-type DRG neurons expressing EGFP vector or the SARM1-CDN and of SARM1 KO neurons expressing EGFP vector after transection. DI ranges from 0 (completely intact) to 1 (completely fragmented). Data are presented as mean ± SEM, tested with a two-way ANOVA, which shows significant main effects of group F(2,9) = 2,710, P
Figure Legend Snippet: SARM1-CDN potently inhibits wild-type SARM1 function. (A) Degeneration of wild-type DRG neurons expressing EGFP vector or the SARM1-CDN and of SARM1 KO neurons expressing EGFP vector after transection. DI ranges from 0 (completely intact) to 1 (completely fragmented). Data are presented as mean ± SEM, tested with a two-way ANOVA, which shows significant main effects of group F(2,9) = 2,710, P

Techniques Used: Expressing, Plasmid Preparation

3) Product Images from "Control of siRNA expression using the Cre-loxP recombination system"

Article Title: Control of siRNA expression using the Cre-loxP recombination system

Journal: Nucleic Acids Research

doi: 10.1093/nar/gnh061

( A ) Luciferase activity due to iGL3BCre-On. Lane 1, negative control (d.m., dialysis medium); lane 2, pU6 control and 4.5 µg/ml TAT-NLS-EGFP (EGFP); lane 3, pU6 control and 4.5 µg/ml TAT-NLS-Cre (Cre); lane 4, pU6iRed and dialysis medium; lane 5, iGL3B and dialysis medium; lane 6, iGL3B loxP and dialysis medium; lane 7, iGL3BCre-On and dialysis medium; lane 8, iGL3BCre-On and residual E.coli lysate (lys); lane 9, iGL3BCre-On and 4.5 µg/ml TAT-NLS-EGFP; lane 10, iGL3BCre-On and 4.5 µg/ml TAT-NLS-Cre. Compared with the result for the negative control (pU6 control and incubation with dialysis medium), the luciferase activity in the cells transfected with iGL3BCre-On and incubated with TAT-NLS-Cre was suppressed by ∼70%, while incubation with dialysis medium, with TAT-NLS-EGFP or with residual E.coli lysate did not suppress luciferase activity. ( B ) Firefly and Renilla luciferase activities of the results shown in (A). The activity of Renilla was not affected by the addition of recombinant protein, dialysis medium or residual E.coli lysate. ( C ) The extent of suppression of luciferase activity decreased with decreases in the concentration of TAT-NLS-Cre, while incubation with TAT-NLS-EGFP at various concentrations failed to have any effect on luciferase activity, which was the same as the control (incubated with dialysis medium). The results were calculated by normalizing the activity due to firefly luciferase to that due to Renilla luciferase. Then the results were calculated as percentages of the control value. Each bar indicates an average value and vertical bars indicate standard errors of triplicate assays.
Figure Legend Snippet: ( A ) Luciferase activity due to iGL3BCre-On. Lane 1, negative control (d.m., dialysis medium); lane 2, pU6 control and 4.5 µg/ml TAT-NLS-EGFP (EGFP); lane 3, pU6 control and 4.5 µg/ml TAT-NLS-Cre (Cre); lane 4, pU6iRed and dialysis medium; lane 5, iGL3B and dialysis medium; lane 6, iGL3B loxP and dialysis medium; lane 7, iGL3BCre-On and dialysis medium; lane 8, iGL3BCre-On and residual E.coli lysate (lys); lane 9, iGL3BCre-On and 4.5 µg/ml TAT-NLS-EGFP; lane 10, iGL3BCre-On and 4.5 µg/ml TAT-NLS-Cre. Compared with the result for the negative control (pU6 control and incubation with dialysis medium), the luciferase activity in the cells transfected with iGL3BCre-On and incubated with TAT-NLS-Cre was suppressed by ∼70%, while incubation with dialysis medium, with TAT-NLS-EGFP or with residual E.coli lysate did not suppress luciferase activity. ( B ) Firefly and Renilla luciferase activities of the results shown in (A). The activity of Renilla was not affected by the addition of recombinant protein, dialysis medium or residual E.coli lysate. ( C ) The extent of suppression of luciferase activity decreased with decreases in the concentration of TAT-NLS-Cre, while incubation with TAT-NLS-EGFP at various concentrations failed to have any effect on luciferase activity, which was the same as the control (incubated with dialysis medium). The results were calculated by normalizing the activity due to firefly luciferase to that due to Renilla luciferase. Then the results were calculated as percentages of the control value. Each bar indicates an average value and vertical bars indicate standard errors of triplicate assays.

Techniques Used: Luciferase, Activity Assay, Negative Control, Incubation, Transfection, Recombinant, Concentration Assay

4) Product Images from "Identification of a multienzyme complex for glucose metabolism in living cells"

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M117.783050

No colocalization of PFKL with other cellular bodies in cells. A–I , EGFP-G3BP ( A ), GFP170* ( D ), and GFP250 ( G ) were cotransfected with PFKL-mOFP ( B , E , and H , respectively) in Hs578T cells. Green channels correspond to the EGFP-fusion constructs, whereas red channels correspond to PFKL-mOFP in the merged images ( C , F , and I ). PFKL-mEGFP formed spatially distinct cellular bodies apart from stress granules and aggresomes. J and K , PFKL-mEGFP and mOFP-tagged formylglycinamidine ribonucleotide synthase were cotransfected into HeLa cells. These two proteins, representing the metabolic complexes in glucose metabolism and de novo purine biosynthesis ( i.e. the purinosome), respectively, do not colocalize in cells ( J ). A random representative region in J was zoomed in for clarification ( K ). Scale bars , 10 μm, unless otherwise indicated.
Figure Legend Snippet: No colocalization of PFKL with other cellular bodies in cells. A–I , EGFP-G3BP ( A ), GFP170* ( D ), and GFP250 ( G ) were cotransfected with PFKL-mOFP ( B , E , and H , respectively) in Hs578T cells. Green channels correspond to the EGFP-fusion constructs, whereas red channels correspond to PFKL-mOFP in the merged images ( C , F , and I ). PFKL-mEGFP formed spatially distinct cellular bodies apart from stress granules and aggresomes. J and K , PFKL-mEGFP and mOFP-tagged formylglycinamidine ribonucleotide synthase were cotransfected into HeLa cells. These two proteins, representing the metabolic complexes in glucose metabolism and de novo purine biosynthesis ( i.e. the purinosome), respectively, do not colocalize in cells ( J ). A random representative region in J was zoomed in for clarification ( K ). Scale bars , 10 μm, unless otherwise indicated.

Techniques Used: Construct, Clarification Assay

5) Product Images from "Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1"

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1

Journal: Antioxidants & Redox Signaling

doi: 10.1089/ars.2011.4264

PmTrx binds to WSSV IE1 in oxidatively stressed Sf9 cells. The upper panel shows the coimmunoprecipitation of the V5-tagged EGFP-PmTrx fusion protein with the Flag-tagged WSSV IE1. Sf9 cells were cotransfected with plasmids containing EGFP-PmTrx-V5, IE1-Flag,
Figure Legend Snippet: PmTrx binds to WSSV IE1 in oxidatively stressed Sf9 cells. The upper panel shows the coimmunoprecipitation of the V5-tagged EGFP-PmTrx fusion protein with the Flag-tagged WSSV IE1. Sf9 cells were cotransfected with plasmids containing EGFP-PmTrx-V5, IE1-Flag,

Techniques Used:

Related Articles

Clone Assay:

Article Title: ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays
Article Snippet: .. EGFP sequence of pEGFP-N3 (Clontech) was replaced with 2xFLAG (DYKDDDDK), resulting in 2FLAG-N3 vector, which was used as a backbone for cloning myo1c coding sequence at Xho I and Bam HI restriction sites, resulting in myo1c-2FLAG-N3. .. GFP in Hsp47-GFP-RDEL was replaced by mCherry by using PCR and subcloning into Bam HI-Not I sites, creating Hsp47-mCherry.

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells
Article Snippet: .. Subsequently, the genes were cloned into either a pmEGFP-N1 plasmid, which possesses an A206K mutation in the EGFP sequence of pEGFP-N1 (Clontech) to produce mEGFP ( ) or the pmOFP-N1 ( ) plasmid expressing monomeric orange fluorescent protein (mOFP). .. The resulting cloned plasmids were confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz).

Article Title: Fat-specific Protein 27 Regulates Storage of Triacylglycerol *Fat-specific Protein 27 Regulates Storage of Triacylglycerol * S⃞
Article Snippet: .. Plasmids and Transfections —pEGFP-FSP27 was created by cloning FSP27 cDNA with 5′ EcoRI and 3′ BamHI sites upstream and in-frame with the eGFP sequence in pEGFP-N1 (Clontech). ..

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

Transfection:

Article Title: Fat-specific Protein 27 Regulates Storage of Triacylglycerol *Fat-specific Protein 27 Regulates Storage of Triacylglycerol * S⃞
Article Snippet: .. Plasmids and Transfections —pEGFP-FSP27 was created by cloning FSP27 cDNA with 5′ EcoRI and 3′ BamHI sites upstream and in-frame with the eGFP sequence in pEGFP-N1 (Clontech). ..

Amplification:

Article Title: The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins
Article Snippet: .. To generate proteins fused to enhanced green fluorescent protein (EGFP) that are expressed in bacteria, the EGFP sequence was first amplified by PCR from the pEGFP-C1 vector (Clontech, Palo Alto, CA) with primers that introduced 5′ NheI and 3′ EcoRI restriction sites. .. The PCR product was digested with NheI and EcoRI and subsequently cloned into the corresponding restriction sites in the pET21b+ vector (Novagen, Madison, WI) that introduces a His6 tag at the C-terminal end of the protein.

Mutagenesis:

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells
Article Snippet: .. Subsequently, the genes were cloned into either a pmEGFP-N1 plasmid, which possesses an A206K mutation in the EGFP sequence of pEGFP-N1 (Clontech) to produce mEGFP ( ) or the pmOFP-N1 ( ) plasmid expressing monomeric orange fluorescent protein (mOFP). .. The resulting cloned plasmids were confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz).

Isolation:

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

Construct:

Article Title: Gene therapy targeting SARM1 blocks pathological axon degeneration in mice
Article Snippet: .. AAV constructs and virus injections AAV vector expressing EGFP under control of the human synapsin promoter was obtained from Addgene (gift from Bryan Roth, School of Medicine at the University of North Carolina at Chapel Hill, NC; Addgene #50465) and used as EGFP vector control (Addgene viral prep #50465-AAV8). pAAV-hSyn-EGFP (Addgene #50465) was cut with BamHI and NcoI, and SARM1-K193R/H194A/H685A (SARM1-CDN) was inserted between the synapsin promoter and EGFP sequence using inFusion (Clontech) system. .. AAV8-hSYN-SARM1-CDN-EGFP was generated by the viral vector core of the Hope Center for Neurological Disorders at Washington University in St. Louis.

Article Title: Control of siRNA expression using the Cre-loxP recombination system
Article Snippet: .. To construct this vector, we first generated a NLS–EGFP fragment by amplifying the EGFP sequence from pEGFP-c2 vector (Clontech, Palo Alto, CA) with a primer containing NLS sequence and inserted it at the NheI and NotI sites of pTriEx-3 Hygro-TAT. .. The plasmid pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and expression of fusion proteins was induced by the addition to the culture medium of isopropyl β- d -thiogalactoside (IPTG).

Sequencing:

Article Title: Gene therapy targeting SARM1 blocks pathological axon degeneration in mice
Article Snippet: .. AAV constructs and virus injections AAV vector expressing EGFP under control of the human synapsin promoter was obtained from Addgene (gift from Bryan Roth, School of Medicine at the University of North Carolina at Chapel Hill, NC; Addgene #50465) and used as EGFP vector control (Addgene viral prep #50465-AAV8). pAAV-hSyn-EGFP (Addgene #50465) was cut with BamHI and NcoI, and SARM1-K193R/H194A/H685A (SARM1-CDN) was inserted between the synapsin promoter and EGFP sequence using inFusion (Clontech) system. .. AAV8-hSYN-SARM1-CDN-EGFP was generated by the viral vector core of the Hope Center for Neurological Disorders at Washington University in St. Louis.

Article Title: ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays
Article Snippet: .. EGFP sequence of pEGFP-N3 (Clontech) was replaced with 2xFLAG (DYKDDDDK), resulting in 2FLAG-N3 vector, which was used as a backbone for cloning myo1c coding sequence at Xho I and Bam HI restriction sites, resulting in myo1c-2FLAG-N3. .. GFP in Hsp47-GFP-RDEL was replaced by mCherry by using PCR and subcloning into Bam HI-Not I sites, creating Hsp47-mCherry.

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells
Article Snippet: .. Subsequently, the genes were cloned into either a pmEGFP-N1 plasmid, which possesses an A206K mutation in the EGFP sequence of pEGFP-N1 (Clontech) to produce mEGFP ( ) or the pmOFP-N1 ( ) plasmid expressing monomeric orange fluorescent protein (mOFP). .. The resulting cloned plasmids were confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz).

Article Title: Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription
Article Snippet: .. The latter plasmid was made by amplifying the EGFP sequence from pEGFP-C1 (Clontech, Leusden, The Netherlands) using primers 5′-CG CTCGAG TATGGTGAGCAAGGGCG-3′ ( XhoI site underlined) and 5′-GC GGGCCC TCCTTGTACAGCTCGTC-3′ ( ApaI site underlined). .. The XhoI / ApaI fragment was inserted in the pLenti6/V5-DEST vector (Invitrogen, Gent, Belgium) to generate COOH-terminal fusion proteins after Gateway-mediated recombination.

Article Title: Control of siRNA expression using the Cre-loxP recombination system
Article Snippet: .. To construct this vector, we first generated a NLS–EGFP fragment by amplifying the EGFP sequence from pEGFP-c2 vector (Clontech, Palo Alto, CA) with a primer containing NLS sequence and inserted it at the NheI and NotI sites of pTriEx-3 Hygro-TAT. .. The plasmid pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and expression of fusion proteins was induced by the addition to the culture medium of isopropyl β- d -thiogalactoside (IPTG).

Article Title: The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins
Article Snippet: .. To generate proteins fused to enhanced green fluorescent protein (EGFP) that are expressed in bacteria, the EGFP sequence was first amplified by PCR from the pEGFP-C1 vector (Clontech, Palo Alto, CA) with primers that introduced 5′ NheI and 3′ EcoRI restriction sites. .. The PCR product was digested with NheI and EcoRI and subsequently cloned into the corresponding restriction sites in the pET21b+ vector (Novagen, Madison, WI) that introduces a His6 tag at the C-terminal end of the protein.

Article Title: Fat-specific Protein 27 Regulates Storage of Triacylglycerol *Fat-specific Protein 27 Regulates Storage of Triacylglycerol * S⃞
Article Snippet: .. Plasmids and Transfections —pEGFP-FSP27 was created by cloning FSP27 cDNA with 5′ EcoRI and 3′ BamHI sites upstream and in-frame with the eGFP sequence in pEGFP-N1 (Clontech). ..

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

Generated:

Article Title: Control of siRNA expression using the Cre-loxP recombination system
Article Snippet: .. To construct this vector, we first generated a NLS–EGFP fragment by amplifying the EGFP sequence from pEGFP-c2 vector (Clontech, Palo Alto, CA) with a primer containing NLS sequence and inserted it at the NheI and NotI sites of pTriEx-3 Hygro-TAT. .. The plasmid pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and expression of fusion proteins was induced by the addition to the culture medium of isopropyl β- d -thiogalactoside (IPTG).

Expressing:

Article Title: Gene therapy targeting SARM1 blocks pathological axon degeneration in mice
Article Snippet: .. AAV constructs and virus injections AAV vector expressing EGFP under control of the human synapsin promoter was obtained from Addgene (gift from Bryan Roth, School of Medicine at the University of North Carolina at Chapel Hill, NC; Addgene #50465) and used as EGFP vector control (Addgene viral prep #50465-AAV8). pAAV-hSyn-EGFP (Addgene #50465) was cut with BamHI and NcoI, and SARM1-K193R/H194A/H685A (SARM1-CDN) was inserted between the synapsin promoter and EGFP sequence using inFusion (Clontech) system. .. AAV8-hSYN-SARM1-CDN-EGFP was generated by the viral vector core of the Hope Center for Neurological Disorders at Washington University in St. Louis.

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells
Article Snippet: .. Subsequently, the genes were cloned into either a pmEGFP-N1 plasmid, which possesses an A206K mutation in the EGFP sequence of pEGFP-N1 (Clontech) to produce mEGFP ( ) or the pmOFP-N1 ( ) plasmid expressing monomeric orange fluorescent protein (mOFP). .. The resulting cloned plasmids were confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz).

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

Polymerase Chain Reaction:

Article Title: The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins
Article Snippet: .. To generate proteins fused to enhanced green fluorescent protein (EGFP) that are expressed in bacteria, the EGFP sequence was first amplified by PCR from the pEGFP-C1 vector (Clontech, Palo Alto, CA) with primers that introduced 5′ NheI and 3′ EcoRI restriction sites. .. The PCR product was digested with NheI and EcoRI and subsequently cloned into the corresponding restriction sites in the pET21b+ vector (Novagen, Madison, WI) that introduces a His6 tag at the C-terminal end of the protein.

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

Plasmid Preparation:

Article Title: Gene therapy targeting SARM1 blocks pathological axon degeneration in mice
Article Snippet: .. AAV constructs and virus injections AAV vector expressing EGFP under control of the human synapsin promoter was obtained from Addgene (gift from Bryan Roth, School of Medicine at the University of North Carolina at Chapel Hill, NC; Addgene #50465) and used as EGFP vector control (Addgene viral prep #50465-AAV8). pAAV-hSyn-EGFP (Addgene #50465) was cut with BamHI and NcoI, and SARM1-K193R/H194A/H685A (SARM1-CDN) was inserted between the synapsin promoter and EGFP sequence using inFusion (Clontech) system. .. AAV8-hSYN-SARM1-CDN-EGFP was generated by the viral vector core of the Hope Center for Neurological Disorders at Washington University in St. Louis.

Article Title: ER sheet persistence is coupled to myosin 1c–regulated dynamic actin filament arrays
Article Snippet: .. EGFP sequence of pEGFP-N3 (Clontech) was replaced with 2xFLAG (DYKDDDDK), resulting in 2FLAG-N3 vector, which was used as a backbone for cloning myo1c coding sequence at Xho I and Bam HI restriction sites, resulting in myo1c-2FLAG-N3. .. GFP in Hsp47-GFP-RDEL was replaced by mCherry by using PCR and subcloning into Bam HI-Not I sites, creating Hsp47-mCherry.

Article Title: Identification of a multienzyme complex for glucose metabolism in living cells
Article Snippet: .. Subsequently, the genes were cloned into either a pmEGFP-N1 plasmid, which possesses an A206K mutation in the EGFP sequence of pEGFP-N1 (Clontech) to produce mEGFP ( ) or the pmOFP-N1 ( ) plasmid expressing monomeric orange fluorescent protein (mOFP). .. The resulting cloned plasmids were confirmed by restriction enzyme digestions and DNA sequencing (GeneWiz).

Article Title: Protocadherin-1 binds to SMAD3 and suppresses TGF-β1-induced gene transcription
Article Snippet: .. The latter plasmid was made by amplifying the EGFP sequence from pEGFP-C1 (Clontech, Leusden, The Netherlands) using primers 5′-CG CTCGAG TATGGTGAGCAAGGGCG-3′ ( XhoI site underlined) and 5′-GC GGGCCC TCCTTGTACAGCTCGTC-3′ ( ApaI site underlined). .. The XhoI / ApaI fragment was inserted in the pLenti6/V5-DEST vector (Invitrogen, Gent, Belgium) to generate COOH-terminal fusion proteins after Gateway-mediated recombination.

Article Title: Control of siRNA expression using the Cre-loxP recombination system
Article Snippet: .. To construct this vector, we first generated a NLS–EGFP fragment by amplifying the EGFP sequence from pEGFP-c2 vector (Clontech, Palo Alto, CA) with a primer containing NLS sequence and inserted it at the NheI and NotI sites of pTriEx-3 Hygro-TAT. .. The plasmid pTriEx-3 Hygro-TAT-NLS-Cre or pTriEx-3 Hygro-TAT-NLS-EGFP was introduced into E.coli strain RosettaBlue(DE3)pLacI (Novagen) and expression of fusion proteins was induced by the addition to the culture medium of isopropyl β- d -thiogalactoside (IPTG).

Article Title: The Bovine Immunodeficiency Virus Rev Protein: Identification of a Novel Nuclear Import Pathway and Nuclear Export Signal among Retroviral Rev/Rev-Like Proteins
Article Snippet: .. To generate proteins fused to enhanced green fluorescent protein (EGFP) that are expressed in bacteria, the EGFP sequence was first amplified by PCR from the pEGFP-C1 vector (Clontech, Palo Alto, CA) with primers that introduced 5′ NheI and 3′ EcoRI restriction sites. .. The PCR product was digested with NheI and EcoRI and subsequently cloned into the corresponding restriction sites in the pET21b+ vector (Novagen, Madison, WI) that introduces a His6 tag at the C-terminal end of the protein.

Article Title: Penaeus monodon Thioredoxin Restores the DNA Binding Activity of Oxidized White Spot Syndrome Virus IE1
Article Snippet: .. Expression of PmTrx was slightly more complicated: because the PmTrx coding regions are so short, the EGFP sequence was isolated from a commercial plasmid pEGFP-N1 (Clontech) by PCR and cloned into the pDHsp70-V5-His vector to produce pDHsp70-EGFP-V5-His. .. The V5-tagged EGFP-PmTrx expression plasmid pDHsp70-EGFP-PmTrx-V5-His was then constructed by PCR using pET28b-rTrx as a template.

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    TaKaRa aid egfp fusion sequences
    Expression level of <t>EGFP-tagged</t> <t>AID</t> mutant proteins correlates with the specific cell compartment in which they reside. (A) Schematic representation of mutations affecting either the NLS (mutNLS, V18S, R19V, according to Shinkura et al. [ 13 ], with numbers indicating amino acid positions within the AID protein) or the NES (mutNES, L189A, F193A, and L196A), with both domains being represented as black boxes. (B) FACS analysis of the tetracycline-inducible clones, with values of the MFI of the EGFP-positive population. By comparison with endogenous AID expression levels in BL2 (corresponding to a MFI value of 15 in the KI clone [ Fig. 1 C ]), AID overexpression in EGFP-positive cells can be estimated at ∼4 for mutNES-AID, 24 for WT-AID, and 90 for mutNLS-AID constructs. (C) Confocal images of EGFP signals from inducible clones, with or without 2-h LMB treatment, are shown together with nuclear staining with propidium iodide (PI) of the same field. Clones with a lower percentage of inducible expression are shown, as indicated by the presence of EGFP-negative cells in the field, a shut-off of expression regularly observed in clones kept in culture in induced conditions. Bars, 20 μm.
    Aid Egfp Fusion Sequences, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa sequence encoding egfp
    Transfected but non-apoptotic cells. A fraction of cells exhibits weak and rather uniform fluorescence when transfected with <t>pcDNA3-C1D(EGFP).</t> Cells of this type can be distinguished because exhibiting undisturbed morphology, and much longer exposure times are required to capture 510 nm fluorescence mode images (2–3 seconds). Left image: phase contrast mode; right image C1D-EGFP fluorescence (510 nm). Bar represents 10 μm.
    Sequence Encoding Egfp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ires egfp sequence
    ( A and C ) <t>EGFP</t> expression, indicated by arrows, in mice infused with P CamkIIα - <t>Sept5-IRES-EGFP</t> into the hippocampus (A) and amygdala (C). Because Sept5 staining does not distinguish endogenous Sept5 and exogenously expressed Sept5, EGFP was used
    Ires Egfp Sequence, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Expression level of EGFP-tagged AID mutant proteins correlates with the specific cell compartment in which they reside. (A) Schematic representation of mutations affecting either the NLS (mutNLS, V18S, R19V, according to Shinkura et al. [ 13 ], with numbers indicating amino acid positions within the AID protein) or the NES (mutNES, L189A, F193A, and L196A), with both domains being represented as black boxes. (B) FACS analysis of the tetracycline-inducible clones, with values of the MFI of the EGFP-positive population. By comparison with endogenous AID expression levels in BL2 (corresponding to a MFI value of 15 in the KI clone [ Fig. 1 C ]), AID overexpression in EGFP-positive cells can be estimated at ∼4 for mutNES-AID, 24 for WT-AID, and 90 for mutNLS-AID constructs. (C) Confocal images of EGFP signals from inducible clones, with or without 2-h LMB treatment, are shown together with nuclear staining with propidium iodide (PI) of the same field. Clones with a lower percentage of inducible expression are shown, as indicated by the presence of EGFP-negative cells in the field, a shut-off of expression regularly observed in clones kept in culture in induced conditions. Bars, 20 μm.

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: Expression level of EGFP-tagged AID mutant proteins correlates with the specific cell compartment in which they reside. (A) Schematic representation of mutations affecting either the NLS (mutNLS, V18S, R19V, according to Shinkura et al. [ 13 ], with numbers indicating amino acid positions within the AID protein) or the NES (mutNES, L189A, F193A, and L196A), with both domains being represented as black boxes. (B) FACS analysis of the tetracycline-inducible clones, with values of the MFI of the EGFP-positive population. By comparison with endogenous AID expression levels in BL2 (corresponding to a MFI value of 15 in the KI clone [ Fig. 1 C ]), AID overexpression in EGFP-positive cells can be estimated at ∼4 for mutNES-AID, 24 for WT-AID, and 90 for mutNLS-AID constructs. (C) Confocal images of EGFP signals from inducible clones, with or without 2-h LMB treatment, are shown together with nuclear staining with propidium iodide (PI) of the same field. Clones with a lower percentage of inducible expression are shown, as indicated by the presence of EGFP-negative cells in the field, a shut-off of expression regularly observed in clones kept in culture in induced conditions. Bars, 20 μm.

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Expressing, Mutagenesis, FACS, Clone Assay, Over Expression, Construct, Staining

    Nuclear destabilization of AID is not restricted to B cells. (A) Human 293T cells transfected with WT-AID-HA–expressing vector were incubated for 6 h with both LMB and MG132, or left untreated, as indicated. Immunoprecipitation was performed using agarose-conjugated anti-HA antibodies, and SDS-PAGE–fractionated protein extracts were analyzed by Western blot with anti-AID antibodies. Migration position of AID-HA is indicated. (B) 293T cells stably transfected with a WT-AID-EGFP–expressing vector were incubated with cycloheximide (CHX; 20 or 50 μg/ml) and LMB, either separately or in combination, and the decay of the protein was followed by the decrease in fluorescence intensity. The percentage of initial fluorescence is plotted at various time points after addition of the inhibitors of protein synthesis and/or protein nuclear export.

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: Nuclear destabilization of AID is not restricted to B cells. (A) Human 293T cells transfected with WT-AID-HA–expressing vector were incubated for 6 h with both LMB and MG132, or left untreated, as indicated. Immunoprecipitation was performed using agarose-conjugated anti-HA antibodies, and SDS-PAGE–fractionated protein extracts were analyzed by Western blot with anti-AID antibodies. Migration position of AID-HA is indicated. (B) 293T cells stably transfected with a WT-AID-EGFP–expressing vector were incubated with cycloheximide (CHX; 20 or 50 μg/ml) and LMB, either separately or in combination, and the decay of the protein was followed by the decrease in fluorescence intensity. The percentage of initial fluorescence is plotted at various time points after addition of the inhibitors of protein synthesis and/or protein nuclear export.

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Immunoprecipitation, SDS Page, Western Blot, Migration, Stable Transfection, Fluorescence

    Lysine mutants of AID are polyubiquitinated in BL2. (A) Schematic presentation of the human AID protein sequence along with the positions of the lysine residues mutated in the various constructs. (B) BL2 clones expressing either WT-AID-EGFP (sample 1, WT), or a lysineless mutant of AID (Kzero-AID-EGFP; sample 2, Kzero) were incubated with the indicated inhibitors for 5 h. Protein extracts were analyzed by immunoblotting with anti-AID. PARP (116 kD) was used as loading control. (C) BL2 cell clones stably expressing either WT-AID-EGFP (WT), mutNES-AID-EGFP (mutNES), a lysineless mutant of AID (Kzero), or the four other AID-EGFP lysine mutants depicted in A were transiently transfected with an HA-tagged ubiquitin-expressing vector and either left untreated (top) or incubated with both LMB and MG132 for 5 h (bottom). Denatured lysates were immunoprecipitated with agarose-conjugated anti-HA antibodies and analyzed by Western blotting using anti-EGFP antibody. Migration position of AID-EGFP is indicated. (D) Stable BL2 clones expressing the indicated AID-EGFP lysine mutants, WT-AID-EGFP, or mutNLS-AID-EGFP were incubated with cycloheximide (CHX, 50 μg/ml) and LMB (10 ng/ml). The decay of the protein was followed by the decrease in fluorescence intensity. The percentage of initial fluorescence is plotted at various time points after addition of the drugs. Kzero1 and 2 represent two independent clones. (top) Clones transfected with AID constructs cloned in the pIRES puro expression vector; (bottom) clones transfected with tetracycline-inducible pBI expression vectors.

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: Lysine mutants of AID are polyubiquitinated in BL2. (A) Schematic presentation of the human AID protein sequence along with the positions of the lysine residues mutated in the various constructs. (B) BL2 clones expressing either WT-AID-EGFP (sample 1, WT), or a lysineless mutant of AID (Kzero-AID-EGFP; sample 2, Kzero) were incubated with the indicated inhibitors for 5 h. Protein extracts were analyzed by immunoblotting with anti-AID. PARP (116 kD) was used as loading control. (C) BL2 cell clones stably expressing either WT-AID-EGFP (WT), mutNES-AID-EGFP (mutNES), a lysineless mutant of AID (Kzero), or the four other AID-EGFP lysine mutants depicted in A were transiently transfected with an HA-tagged ubiquitin-expressing vector and either left untreated (top) or incubated with both LMB and MG132 for 5 h (bottom). Denatured lysates were immunoprecipitated with agarose-conjugated anti-HA antibodies and analyzed by Western blotting using anti-EGFP antibody. Migration position of AID-EGFP is indicated. (D) Stable BL2 clones expressing the indicated AID-EGFP lysine mutants, WT-AID-EGFP, or mutNLS-AID-EGFP were incubated with cycloheximide (CHX, 50 μg/ml) and LMB (10 ng/ml). The decay of the protein was followed by the decrease in fluorescence intensity. The percentage of initial fluorescence is plotted at various time points after addition of the drugs. Kzero1 and 2 represent two independent clones. (top) Clones transfected with AID constructs cloned in the pIRES puro expression vector; (bottom) clones transfected with tetracycline-inducible pBI expression vectors.

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Sequencing, Construct, Clone Assay, Expressing, Mutagenesis, Incubation, Stable Transfection, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Migration, Fluorescence

    The half-life of AID differs according to its subcellular localization. (A and B) BL2 clones expressing WT-AID-EGFP, MutNES-AID-EGFP, or MutNLS-AID-EGFP were metabolically labeled for 1 h with [ 35 S]-labeled cysteine and methionine and chased for the indicated length of time. AID-EGFP was immunoprecipitated from cell lysates with anti-EGFP antibody (or anti-AID for the endogenous protein) and subjected to SDS-PAGE and autoradiography. (C) Immunoprecipitated MutNES-AID-EGFP was transferred into nitrocellulose and probed with a monoclonal HRP-conjugated anti-EGFP antibody to control for immunoprecipitation efficiency. (D) Densitometry analysis of autoradiographs of pulse-chase immunoprecipitated products, expressed as percentage of initial labeling. The data shown are the mean of the results from two experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: The half-life of AID differs according to its subcellular localization. (A and B) BL2 clones expressing WT-AID-EGFP, MutNES-AID-EGFP, or MutNLS-AID-EGFP were metabolically labeled for 1 h with [ 35 S]-labeled cysteine and methionine and chased for the indicated length of time. AID-EGFP was immunoprecipitated from cell lysates with anti-EGFP antibody (or anti-AID for the endogenous protein) and subjected to SDS-PAGE and autoradiography. (C) Immunoprecipitated MutNES-AID-EGFP was transferred into nitrocellulose and probed with a monoclonal HRP-conjugated anti-EGFP antibody to control for immunoprecipitation efficiency. (D) Densitometry analysis of autoradiographs of pulse-chase immunoprecipitated products, expressed as percentage of initial labeling. The data shown are the mean of the results from two experiments.

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Clone Assay, Expressing, Metabolic Labelling, Labeling, Immunoprecipitation, SDS Page, Autoradiography, Pulse Chase

    Ubiquitination of AID and AID-EGFP requires both nuclear localization and proteasome inhibition. (A) AID-EGFP knocked-in BL2 cells, expressing both AID and AID-EGFP under its endogenous promoter, were incubated with MG132 to inhibit proteasome activity. Cell lysates from the indicated time points were analyzed by immunoblotting using anti-AID monoclonal antibodies. (B) BL2 cells expressing both endogenous AID and MutNES-AID-EGFP were incubated for 5 h with LMB and/or MG132, as indicated. AID status was analyzed as described in A. Poly(ADP-ribose) polymerase (PARP; 116 kD) was used as loading control. (C) BL2 cells were incubated with LMB, MG132, or both, as indicated. For each condition, cytoplasmic (C) and nuclear (N) protein extracts were prepared as described in Materials and methods and analyzed by immunoblotting, with monoclonal anti-AID antibodies. Anti-PARP antibody was used as a nuclear protein control. (D) A BL2 cell line expressing WT-AID-HA was incubated with LMB or MG132, or both, for 5 h. After treatment, cell lysates were denatured and immunoprecipitated with agarose-conjugated anti-HA antibodies before SDS-PAGE separation and probing with anti-AID (top) or antiubiquitin antibodies (bottom). MW markers and migration positions of AID-HA are indicated. (E) AID-EGFP knocked-in BL2 cells were transfected with an HA-tagged ubiquitin-expressing vector and either incubated or not with LMB and MG132 for 5 h. Immunoprecipitation was performed 16 h after transfection, as described in D. Western blot analysis was performed using anti-AID antibodies. Lanes: C, extract before immunoprecipitation (1/10th of total extract); IP, immunoprecipitated proteins.

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: Ubiquitination of AID and AID-EGFP requires both nuclear localization and proteasome inhibition. (A) AID-EGFP knocked-in BL2 cells, expressing both AID and AID-EGFP under its endogenous promoter, were incubated with MG132 to inhibit proteasome activity. Cell lysates from the indicated time points were analyzed by immunoblotting using anti-AID monoclonal antibodies. (B) BL2 cells expressing both endogenous AID and MutNES-AID-EGFP were incubated for 5 h with LMB and/or MG132, as indicated. AID status was analyzed as described in A. Poly(ADP-ribose) polymerase (PARP; 116 kD) was used as loading control. (C) BL2 cells were incubated with LMB, MG132, or both, as indicated. For each condition, cytoplasmic (C) and nuclear (N) protein extracts were prepared as described in Materials and methods and analyzed by immunoblotting, with monoclonal anti-AID antibodies. Anti-PARP antibody was used as a nuclear protein control. (D) A BL2 cell line expressing WT-AID-HA was incubated with LMB or MG132, or both, for 5 h. After treatment, cell lysates were denatured and immunoprecipitated with agarose-conjugated anti-HA antibodies before SDS-PAGE separation and probing with anti-AID (top) or antiubiquitin antibodies (bottom). MW markers and migration positions of AID-HA are indicated. (E) AID-EGFP knocked-in BL2 cells were transfected with an HA-tagged ubiquitin-expressing vector and either incubated or not with LMB and MG132 for 5 h. Immunoprecipitation was performed 16 h after transfection, as described in D. Western blot analysis was performed using anti-AID antibodies. Lanes: C, extract before immunoprecipitation (1/10th of total extract); IP, immunoprecipitated proteins.

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Inhibition, Expressing, Incubation, Activity Assay, Immunoprecipitation, SDS Page, Migration, Transfection, Plasmid Preparation, Western Blot

    Variation of AID expression during the cell cycle. (A) EGFP KI at the AICDA locus in BL2 cells. The AID–EGFP KI construct includes the EGFP sequence inserted in-frame in exon 5 at the 3′ end of the AID coding region and a hygromycin resistance (hygro R ) gene flanked by loxP sites. Configuration of the targeted AICDA locus is depicted after Cre-mediated excision of the hygro R gene. (B) Expression of AID-EGFP throughout the cell cycle. 48 h after IL-4 addition (10 ng/ml), AID-EGFP KI BL2 cells were fractionated according to their cell cycle status using counterflow elutriation. Collected fractions were stained with propidium iodide and analyzed for both DNA content and AID-EGFP MFI. Data for fractions 12, 30, and 39 are shown in C. (C) Cell cycle analysis and AID-EGFP expression level of the BL2 KI cell line with and without IL-4, and of representative elutriated fractions, corresponding to the different phases of cell cycle: G1 (fr.12), S (fr. 30), and G2/M (fr. 39).

    Journal: The Journal of Experimental Medicine

    Article Title: Proteasomal degradation restricts the nuclear lifespan of AID

    doi: 10.1084/jem.20070950

    Figure Lengend Snippet: Variation of AID expression during the cell cycle. (A) EGFP KI at the AICDA locus in BL2 cells. The AID–EGFP KI construct includes the EGFP sequence inserted in-frame in exon 5 at the 3′ end of the AID coding region and a hygromycin resistance (hygro R ) gene flanked by loxP sites. Configuration of the targeted AICDA locus is depicted after Cre-mediated excision of the hygro R gene. (B) Expression of AID-EGFP throughout the cell cycle. 48 h after IL-4 addition (10 ng/ml), AID-EGFP KI BL2 cells were fractionated according to their cell cycle status using counterflow elutriation. Collected fractions were stained with propidium iodide and analyzed for both DNA content and AID-EGFP MFI. Data for fractions 12, 30, and 39 are shown in C. (C) Cell cycle analysis and AID-EGFP expression level of the BL2 KI cell line with and without IL-4, and of representative elutriated fractions, corresponding to the different phases of cell cycle: G1 (fr.12), S (fr. 30), and G2/M (fr. 39).

    Article Snippet: AID-EGFP fusion sequences were cloned in the pIRESpuro vector or in the tetracycline-inducible pBI expression vector (Clontech Laboratories), in which a blasticidin resistance gene amplified from the pcDNA6/TR plasmid (Invitrogen) was inserted in its AatII site to allow for selection of transfected clones.

    Techniques: Expressing, Construct, Sequencing, Staining, Cell Cycle Assay

    Transfected but non-apoptotic cells. A fraction of cells exhibits weak and rather uniform fluorescence when transfected with pcDNA3-C1D(EGFP). Cells of this type can be distinguished because exhibiting undisturbed morphology, and much longer exposure times are required to capture 510 nm fluorescence mode images (2–3 seconds). Left image: phase contrast mode; right image C1D-EGFP fluorescence (510 nm). Bar represents 10 μm.

    Journal: Cancer Cell International

    Article Title: Proteasome-mediated degradation antagonizes critical levels of the apoptosis-inducing C1D protein

    doi: 10.1186/1475-2867-2-12

    Figure Lengend Snippet: Transfected but non-apoptotic cells. A fraction of cells exhibits weak and rather uniform fluorescence when transfected with pcDNA3-C1D(EGFP). Cells of this type can be distinguished because exhibiting undisturbed morphology, and much longer exposure times are required to capture 510 nm fluorescence mode images (2–3 seconds). Left image: phase contrast mode; right image C1D-EGFP fluorescence (510 nm). Bar represents 10 μm.

    Article Snippet: Briefly, the PCR-amplified sequence encoding the murine C1D protein (X95591) was fused with the sequence encoding EGFP (Clontech) in the pBluescript KS+ vector (Stratagene).

    Techniques: Transfection, Fluorescence

    Distribution of CPXs III and IV in neurons. Neurons transfected with expression vectors encoding either wt CPX I-EGFP, CPX II-EGFP, EGFP-CPX III, EGFP-CPX IV or farnesylation-deficient EGFP-CPX III-C156S, and EGFP-CPX IV-C158S (all green), and immunostained for synaptophysin (red) to mark presynaptic terminals were analyzed by laser scanning confocal microscopy. Wt CPXs III (top left) and IV (middle left) were concentrated in presynaptic structures of axons and colocalized with synaptophysin, whereas mutant CPXs III (top right) and IV (middle right) as well as wt CPXs I (bottom left) and II (bottom right) showed a diffuse axonal distribution with minor (CPXs I and II) or no (mutant CPXs III and IV) presynaptic accumulation. Bars: (low magnification, large images) 50 μm; (high magnification, small images) 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: Structurally and functionally unique complexins at retinal ribbon synapses

    doi: 10.1083/jcb.200502115

    Figure Lengend Snippet: Distribution of CPXs III and IV in neurons. Neurons transfected with expression vectors encoding either wt CPX I-EGFP, CPX II-EGFP, EGFP-CPX III, EGFP-CPX IV or farnesylation-deficient EGFP-CPX III-C156S, and EGFP-CPX IV-C158S (all green), and immunostained for synaptophysin (red) to mark presynaptic terminals were analyzed by laser scanning confocal microscopy. Wt CPXs III (top left) and IV (middle left) were concentrated in presynaptic structures of axons and colocalized with synaptophysin, whereas mutant CPXs III (top right) and IV (middle right) as well as wt CPXs I (bottom left) and II (bottom right) showed a diffuse axonal distribution with minor (CPXs I and II) or no (mutant CPXs III and IV) presynaptic accumulation. Bars: (low magnification, large images) 50 μm; (high magnification, small images) 10 μm.

    Article Snippet: Expression of recombinant proteins in HEK293 cells Mammalian expression vectors encoding full-length wt rCPX I, rCPX II, mCPX III, and mCPX IV with the EGFP sequence attached at their NH2 (CPXs III and IV) or COOH (CPXs I and II) termini were constructed in pEGFP-C1 and pEGFP-N1 (CLONTECH Laboratories, Inc.), respectively.

    Techniques: Transfection, Expressing, Confocal Microscopy, Mutagenesis

    In vivo farnesylation of CPXs III and IV. Transfected HEK293 cells coexpressing a mevalonate transporter and either wt EGFP-CPXs III or IV (CPX III, CPX IV), or mutant EGFP-CPXs III or IV (CPX III-C156S, CPX IV-C158S) were labeled with [ 14 C]mevalonate and harvested. Control cells were only transfected with the mevalonate transporter. Whole cell lysates as well as solubilized and immunoprecipitated proteins were analyzed by Western blotting, using an mAb to EGFP (Western blot), and autoradiography (Autoradiograph). The arrows indicate the EGFP-CPX–fusion proteins. All proteins were expressed and precipitated (bottom) but only wt proteins were 14 C-labeled (top).

    Journal: The Journal of Cell Biology

    Article Title: Structurally and functionally unique complexins at retinal ribbon synapses

    doi: 10.1083/jcb.200502115

    Figure Lengend Snippet: In vivo farnesylation of CPXs III and IV. Transfected HEK293 cells coexpressing a mevalonate transporter and either wt EGFP-CPXs III or IV (CPX III, CPX IV), or mutant EGFP-CPXs III or IV (CPX III-C156S, CPX IV-C158S) were labeled with [ 14 C]mevalonate and harvested. Control cells were only transfected with the mevalonate transporter. Whole cell lysates as well as solubilized and immunoprecipitated proteins were analyzed by Western blotting, using an mAb to EGFP (Western blot), and autoradiography (Autoradiograph). The arrows indicate the EGFP-CPX–fusion proteins. All proteins were expressed and precipitated (bottom) but only wt proteins were 14 C-labeled (top).

    Article Snippet: Expression of recombinant proteins in HEK293 cells Mammalian expression vectors encoding full-length wt rCPX I, rCPX II, mCPX III, and mCPX IV with the EGFP sequence attached at their NH2 (CPXs III and IV) or COOH (CPXs I and II) termini were constructed in pEGFP-C1 and pEGFP-N1 (CLONTECH Laboratories, Inc.), respectively.

    Techniques: In Vivo, Transfection, Mutagenesis, Labeling, Immunoprecipitation, Western Blot, Autoradiography

    ( A and C ) EGFP expression, indicated by arrows, in mice infused with P CamkIIα - Sept5-IRES-EGFP into the hippocampus (A) and amygdala (C). Because Sept5 staining does not distinguish endogenous Sept5 and exogenously expressed Sept5, EGFP was used

    Journal: Human Molecular Genetics

    Article Title: Alterations of social interaction through genetic and environmental manipulation of the 22q11.2 gene Sept5 in the mouse brain

    doi: 10.1093/hmg/dds180

    Figure Lengend Snippet: ( A and C ) EGFP expression, indicated by arrows, in mice infused with P CamkIIα - Sept5-IRES-EGFP into the hippocampus (A) and amygdala (C). Because Sept5 staining does not distinguish endogenous Sept5 and exogenously expressed Sept5, EGFP was used

    Article Snippet: The IRES-EGFP sequence (Clontech, Mountain View, CA, USA) was amplified with primers with attB2/attB3 and introduced into a pDONR-P2R-P3 vector (Invitrogen) by BP reaction.

    Techniques: Expressing, Mouse Assay, Staining