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addgene plasmid 119947 (Addgene inc)

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Addgene inc addgene plasmid 119947
Addgene Plasmid 119947, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/addgene+plasmid+119947/phage+UbiC+tagRFP-T-DDX6+(Plasmid+%23119947)/pmc12511425-402-3-3
Average 93 stars, based on 8 article reviews

addgene plasmid 119947 - by Bioz Stars, 2026-09

93/100 stars

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Over Expression:

Article Title: Ddx61-enriched condensates refine heart regeneration programs.
Article Snippet: Twenty micrograms of protein per lane was run on Bolt 4–12%, Bis-Tris gels (Thermo NW04120) prior to transfer to 0.2 μm PVDF membranes. .. DDX6 overexpression For DDX6 overexpression, Addgene plasmid 119947 (phage UbiC tagRFP-T-DDX6 was a gift from Jeffrey Chao, Addgene plasmid # 119947; http://n2t.net/addgene:119947; RRID:Addgene_119947) was used to generate lentivirus. ..

Article Title: Ddx61-enriched condensates refine heart regeneration programs
Article Snippet: .. For DDX6 overexpression, Addgene plasmid 119947 (phage UbiC tagRFP-T-DDX6 was a gift from Jeffrey Chao, Addgene plasmid # 119947; http://n2t.net/addgene:119947 ; RRID:Addgene_119947) was used to generate lentivirus. ..

Plasmid Preparation:

Article Title: Programmable Control of Spatial Transcriptome in Live Cells and Neurons
Article Snippet: The Dendra2 sequence was amplified from Addgene plasmid 57701. .. For cloning PYL1-fusion proteins, the MAVS(aa510–540) sequence was ordered as gBlock from Integrated DNA Technologies (IDT), the DDX6 sequence was amplified from Addgene plasmid 119947, the G3BP1 sequence was amplified from the cDNA of U2OS cells, the KIFC1(aa125–673) sequence was amplified from Addgene plasmid 120169, the Kif5b(aa1–555) sequence was amplified from Addgene plasmid 120164, the Kif5a(aa1–559) sequence was amplified from Addgene plasmid 120163, the HSF1 sequence was amplified from Addgene plasmid 32538, and the TRF1 sequence was amplified from Addgene plasmid 169449. .. For cloning reporter mRNAs with different GCN4 repeats, 1x, 2x, 3x, and 7x GCN4 fragments were inserted into Addgene plasmid 132413 to replace the original 24xGCN4 sequence, followed by changing the CMV promoter to a PGK promoter.

other:

Article Title: Programmable control of spatial transcriptome in live cells and neurons.
Article Snippet: Spatial RNA organization has a pivotal role in diverse cellular processes and diseases.. However, functional implications of the spatial transcriptome remain largely unexplored due to limited technologies for perturbing endogenous RNA within specific subcellular regions.. Here we present CRISPR-mediated transcriptome organization (CRISPR-TO), a system that harnesses RNA-guided, nuclease-dead dCas13 for programmable control of endogenous RNA localization in live cells.

Cloning:

Sequencing:

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Image Search Results

Figure 1. COPII-mediated cargo transport continues in the absence of Sar1 (A) Cartoon depicting the human SAR1A and SAR1B genomic loci. The positions of the gRNAs used during CRISPR-Cas9 editing (red lines) are highlighted, and the sizes of exons (shown as green boxes) are 1/50 that of introns (shown as black lines). (B) Representative immunoblots of extracts generated from a CRISPR-modiﬁed cell line lack- ing Sar1a and subjected to siRNA-mediated treatments as shown, using antibodies directed against Sar1 and actin. Extracts were generated at the time point indicated following siRNA treat- ment. (C) Quantiﬁcation of the percentage of Sar1 re- maining at different time points following Sar1b siRNA treatment (relative to mock siRNA treat- ment). Error bars represent mean ± SEM (n = 4 biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (D) Spinning disk confocal microscopy was used to image control RPE1 cells and cells lacking Sar1a either in the presence or in the absence of Sar1b, each expressing ss-DsRed following treatment with SLF (50 mM) to induce cargo disaggregation and release from the ER. Repre- sentative time-lapse images are shown (n = 15 cells, each condition; at least three biological replicates). Scale bar, 5 mm. (E and F) Quantiﬁcation of cargo (E, ss-DsRed; F, ManII-SBP-GFP) accumulation within the peri- nuclear region (GM130 positive) in the various mutant backgrounds indicated. Error bars repre- sent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to the 0 min time point). See also Figures S1–S3.

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 1. COPII-mediated cargo transport continues in the absence of Sar1 (A) Cartoon depicting the human SAR1A and SAR1B genomic loci. The positions of the gRNAs used during CRISPR-Cas9 editing (red lines) are highlighted, and the sizes of exons (shown as green boxes) are 1/50 that of introns (shown as black lines). (B) Representative immunoblots of extracts generated from a CRISPR-modiﬁed cell line lack- ing Sar1a and subjected to siRNA-mediated treatments as shown, using antibodies directed against Sar1 and actin. Extracts were generated at the time point indicated following siRNA treat- ment. (C) Quantiﬁcation of the percentage of Sar1 re- maining at different time points following Sar1b siRNA treatment (relative to mock siRNA treat- ment). Error bars represent mean ± SEM (n = 4 biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (D) Spinning disk confocal microscopy was used to image control RPE1 cells and cells lacking Sar1a either in the presence or in the absence of Sar1b, each expressing ss-DsRed following treatment with SLF (50 mM) to induce cargo disaggregation and release from the ER. Repre- sentative time-lapse images are shown (n = 15 cells, each condition; at least three biological replicates). Scale bar, 5 mm. (E and F) Quantiﬁcation of cargo (E, ss-DsRed; F, ManII-SBP-GFP) accumulation within the peri- nuclear region (GM130 positive) in the various mutant backgrounds indicated. Error bars repre- sent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to the 0 min time point). See also Figures S1–S3.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER phage UbiC tagRFP-T-DDX6 Addgene 119947 pTALEN TET Sar1a (H79G) This study N/A pTALEN TET Sar1a (T39N) This study N/A pCMV KDEL-DsRed This study N/A pEYFP-Sec31a Addgene 66613 AICSDP-25:MAP1LC3B-mEGFP Addgene 101783 AICSDP-7:Sec61b mEGFP Addgene 87426 PX458 Sar1a gRNA CRISPR (5’- GATGTAGTGTTGGAACATGT-3’) David Ginsburg N/A PX458 Sar1b gRNA CRISPR (5’-CAATGCCATTGATAGCAGGA-3’) David Ginsburg N/A PX458 Sec23b gRNA CRISPR (5’- GGAACGTGTGGCCTTCCAGC-3’) David Ginsburg N/A Software and algorithms EasyFRAP Rapsomaniki et al., 2012 https://doi.org/10.1093/bioinformatics/bts241 IMARIS Bitplane N/A FIJI Schneider et al., 2012 https://doi.org/10.1038/nmeth.2089

Techniques: CRISPR, Western Blot, Generated, Confocal Microscopy, Control, Expressing, Mutagenesis

Figure 2. Loss of Sar1 alters the distribution of COPII coat subunits but fails to block their ability to co-assemble (A) Genome-edited cells expressing HaloTag- Sec23a and lacking Sar1a (mock transfected) and those depleted of Sar1b for 48 h were imaged live using spinning disk confocal microscopy following labeling with JFX646-HaloTag ligand. Scale bar, 5 mm; inset bar, 2 mm. (B and C) Quantiﬁcation of the number of Sec23a- positive sites in cells lacking Sar1a in the presence and absence of Sar1b is shown at various time points following Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test, relative to mock treatment (B) or the 48 h time point (C). (D) Cells lacking Sar1a, depleted of Sar1b, and co- expressing HaloTag-Sec23a and YFP-Sec31a were imaged live using spinning disk confocal microscopy following labeling using JFX646- HaloTag ligand. Representative zoomed images are shown (n = 10 cells; at least three biological replicates each). Scale bar, 2 mm. (E) The volume distribution of Sec23a-positive structures in the absence of Sar1a is shown following 48 h of mock treatment or Sar1b deple- tion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to mock treatment). (F) Cells lacking Sar1a (mock transfected) or depleted of Sar1b for 72 h were immunostained using antibodies directed against Sec24a and TFG (shown only in insets). Representative confocal images (maximum intensity projections) are shown, and an arrow indicates co-localization of Sec24a and TFG. Scale bar, 5 mm; inset bar, 2 mm. (G–I) HaloTag-Sec23a in cells lacking Sar1a, either in the presence or in the absence of Sar1b, were labeled with JFX646-HaloTag ligand and sub- jected to photobleaching. Fluorescence recovery is depicted for various-sized HaloTag-Sec23a structures (n = 15 cells each; at least three bio- logical replicates each). See also Figures S4–S6.

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 2. Loss of Sar1 alters the distribution of COPII coat subunits but fails to block their ability to co-assemble (A) Genome-edited cells expressing HaloTag- Sec23a and lacking Sar1a (mock transfected) and those depleted of Sar1b for 48 h were imaged live using spinning disk confocal microscopy following labeling with JFX646-HaloTag ligand. Scale bar, 5 mm; inset bar, 2 mm. (B and C) Quantiﬁcation of the number of Sec23a- positive sites in cells lacking Sar1a in the presence and absence of Sar1b is shown at various time points following Sar1b depletion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test, relative to mock treatment (B) or the 48 h time point (C). (D) Cells lacking Sar1a, depleted of Sar1b, and co- expressing HaloTag-Sec23a and YFP-Sec31a were imaged live using spinning disk confocal microscopy following labeling using JFX646- HaloTag ligand. Representative zoomed images are shown (n = 10 cells; at least three biological replicates each). Scale bar, 2 mm. (E) The volume distribution of Sec23a-positive structures in the absence of Sar1a is shown following 48 h of mock treatment or Sar1b deple- tion. Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates). **p < 0.01 and *p < 0.05, calculated using an ANOVA followed by a Tukey post hoc test (relative to mock treatment). (F) Cells lacking Sar1a (mock transfected) or depleted of Sar1b for 72 h were immunostained using antibodies directed against Sec24a and TFG (shown only in insets). Representative confocal images (maximum intensity projections) are shown, and an arrow indicates co-localization of Sec24a and TFG. Scale bar, 5 mm; inset bar, 2 mm. (G–I) HaloTag-Sec23a in cells lacking Sar1a, either in the presence or in the absence of Sar1b, were labeled with JFX646-HaloTag ligand and sub- jected to photobleaching. Fluorescence recovery is depicted for various-sized HaloTag-Sec23a structures (n = 15 cells each; at least three bio- logical replicates each). See also Figures S4–S6.

Techniques: Blocking Assay, Expressing, Transfection, Confocal Microscopy, Labeling, Fluorescence

Figure 3. COPII condensates associate with secretory cargoes that leave the ER in the absence of Sar1 (A) Cells expressing ss-DsRed and lacking both Sar1 isoforms were immunostained using antibodies directed against Sec24a and GM130 following treatment with SLF (50 mM) and imaged using STED microscopy. Representative images are shown (n = 15 cells; at least three biological replicates). Scale bar, 2 mm. (B) Cells expressing EGFP-Sec61b and HaloTag-Sec23a in the absence of Sar1 were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following cargo release and labeling with JFX646-HaloTag ligand. Violin plots show the relative displacement of each marker over time (n = 10 cells; three biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test, compared with Sec61b displacement. (C) Cells lacking both Sar1 isoforms and expressing native EGFP-Sec61b and HaloTag-Sec23a were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following treatment with SLF (50 mM) and labeling with JFX646-HaloTag ligand. Representative images are shown (n = 7 cells; three biological replicates). Arrowheads indicate accumulation of ss-DsRed with COPII condensates, which ultimately move away from their site of origin. Scale bar, 2 mm. (D) Relative ﬂuorescence intensities of cargo (ss-DsRed) and COPII (HaloTag-Sec23a) were measured at ER subdomains over time. Error bars represent mean ± SEM. An asterisk highlights the time point at which cargo and COPII undergo a >1 mm displacement, and all measurements are aligned with respect to this time point (n = 10 cells; three biological replicates). (E) Expression of 43FM-HaloTag-L1CAM was transiently induced in control cells co-expressing Sar1 (H79G) or in cells lacking Sar1a and depleted of Sar1b, each labeled with JFX646-HaloTag ligand, and subjected to photobleaching after treatment with DDS for 60 min. Normalized ﬂuorescence recovery in each case is shown (n = 10 cells each; at least three biological replicates each), and error bars represent mean ± SEM. An asterisk highlights the time point at which individual sites exhibiting elevated 43FM-HaloTag-L1CAM ﬂuorescence were bleached. (F) Cells lacking Sar1a and natively co-expressing EGFP-Sec61b and HaloTag-Sec23a in the presence and absence of Sar1b were transfected with a construct encoding ss-DsRed and imaged as described for (B). The length of time ss-DsRed remained associated with HaloTag-Sec23A prior to undergoing displacement (more than 1 mm) was determined in each case. **p < 0.01, calculated using a t test, compared with cells lacking only Sar1a. See also Figure S7.

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 3. COPII condensates associate with secretory cargoes that leave the ER in the absence of Sar1 (A) Cells expressing ss-DsRed and lacking both Sar1 isoforms were immunostained using antibodies directed against Sec24a and GM130 following treatment with SLF (50 mM) and imaged using STED microscopy. Representative images are shown (n = 15 cells; at least three biological replicates). Scale bar, 2 mm. (B) Cells expressing EGFP-Sec61b and HaloTag-Sec23a in the absence of Sar1 were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following cargo release and labeling with JFX646-HaloTag ligand. Violin plots show the relative displacement of each marker over time (n = 10 cells; three biological replicates). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test, compared with Sec61b displacement. (C) Cells lacking both Sar1 isoforms and expressing native EGFP-Sec61b and HaloTag-Sec23a were transfected with a construct encoding ss-DsRed and imaged live using spinning disk confocal microscopy following treatment with SLF (50 mM) and labeling with JFX646-HaloTag ligand. Representative images are shown (n = 7 cells; three biological replicates). Arrowheads indicate accumulation of ss-DsRed with COPII condensates, which ultimately move away from their site of origin. Scale bar, 2 mm. (D) Relative ﬂuorescence intensities of cargo (ss-DsRed) and COPII (HaloTag-Sec23a) were measured at ER subdomains over time. Error bars represent mean ± SEM. An asterisk highlights the time point at which cargo and COPII undergo a >1 mm displacement, and all measurements are aligned with respect to this time point (n = 10 cells; three biological replicates). (E) Expression of 43FM-HaloTag-L1CAM was transiently induced in control cells co-expressing Sar1 (H79G) or in cells lacking Sar1a and depleted of Sar1b, each labeled with JFX646-HaloTag ligand, and subjected to photobleaching after treatment with DDS for 60 min. Normalized ﬂuorescence recovery in each case is shown (n = 10 cells each; at least three biological replicates each), and error bars represent mean ± SEM. An asterisk highlights the time point at which individual sites exhibiting elevated 43FM-HaloTag-L1CAM ﬂuorescence were bleached. (F) Cells lacking Sar1a and natively co-expressing EGFP-Sec61b and HaloTag-Sec23a in the presence and absence of Sar1b were transfected with a construct encoding ss-DsRed and imaged as described for (B). The length of time ss-DsRed remained associated with HaloTag-Sec23A prior to undergoing displacement (more than 1 mm) was determined in each case. **p < 0.01, calculated using a t test, compared with cells lacking only Sar1a. See also Figure S7.

Techniques: Expressing, Microscopy, Transfection, Construct, Confocal Microscopy, Labeling, Marker, Control

Figure 4. The absence of Sar1 destabilizes ERGIC membranes (A, B, D, E, and F) Cells lacking Sar1a (mock transfected or depleted of Sar1b for 72 h) were immunostained using antibodies directed against Sec16a (A), Tango1 (B), ERGIC-53 (and Sec16a shown in insets, with arrows highlighting their juxtaposed distribution) (D), TFG (E), or COPB1 (and GM130 shown in insets) (F). Representative confocal images (maximum intensity projections) are shown. Scale bars, 5 mm; inset bars, 2 mm. (C) Quantiﬁcation of the fold change in the ﬂuorescence intensities of Sec31a, Sec16a, and Tango1 in the absence of Sar1 (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (G) Representative immunoblots of extracts generated from control and CRISPR-modiﬁed cell lines lacking Sar1a and either in the presence or in the absence of Sar1b, using antibodies directed against COPB1 and actin. (H) Quantiﬁcation of the fold change in COPB1 levels in the absence of Sar1 (relative to mock siRNA treatment of cells lacking only Sar1a). Error bar represents mean ± SEM (n = 4 biological replicates). See also Figure S8.

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 4. The absence of Sar1 destabilizes ERGIC membranes (A, B, D, E, and F) Cells lacking Sar1a (mock transfected or depleted of Sar1b for 72 h) were immunostained using antibodies directed against Sec16a (A), Tango1 (B), ERGIC-53 (and Sec16a shown in insets, with arrows highlighting their juxtaposed distribution) (D), TFG (E), or COPB1 (and GM130 shown in insets) (F). Representative confocal images (maximum intensity projections) are shown. Scale bars, 5 mm; inset bars, 2 mm. (C) Quantiﬁcation of the fold change in the ﬂuorescence intensities of Sec31a, Sec16a, and Tango1 in the absence of Sar1 (relative to mock siRNA treatment). Error bars represent mean ± SEM (n = 15 cells each; at least three biological replicates each). **p < 0.01, calculated using an ANOVA followed by a Tukey post hoc test. (G) Representative immunoblots of extracts generated from control and CRISPR-modiﬁed cell lines lacking Sar1a and either in the presence or in the absence of Sar1b, using antibodies directed against COPB1 and actin. (H) Quantiﬁcation of the fold change in COPB1 levels in the absence of Sar1 (relative to mock siRNA treatment of cells lacking only Sar1a). Error bar represents mean ± SEM (n = 4 biological replicates). See also Figure S8.

Techniques: Transfection, Western Blot, Generated, Control, CRISPR

Figure 5. The absence of Sar1 leads to the formation of COPII condensates that inter- fere with secretory cargo movement through the Golgi (A) Time-lapse spinning disk confocal microscopy was used to image cargo (ss-DsRed) accumulated in the perinuclear region of cells lacking Sar1 following addition of 1,6-hexanediol (top) or 2,5- hexanediol (bottom). Representative images are shown (n = 10 cells; three biological replicates). Scale bar, 5 mm. (B) Quantiﬁcation of cargo (ss-DsRed) remaining within the perinuclear region of cells lacking Sar1 following 30 min of incubation with 1,6-hexanediol or 2,5-hexanediol (relative to the 0 min time point). Error bars represent mean ± SEM (n = 10 cells each; three biological replicates). ***p < 0.001, calculated using a t test, compared with treatment with 2,5-hexanediol. (C) Representative electron micrographs taken within the perinuclear region of high-pressure- frozen cells lacking Sar1a following a mock siRNA treatment or Sar1b depletion. The nuclear enve- lope (NE) is indicated in each image (n = 5 cells each; at least three biological replicates). Scale bar, 500 nm. See also Figure S9.

Journal: Cell reports

Article Title: The Sar1 GTPase is dispensable for COPII-dependent cargo export from the ER.

doi: 10.1016/j.celrep.2023.112635

Figure Lengend Snippet: Figure 5. The absence of Sar1 leads to the formation of COPII condensates that inter- fere with secretory cargo movement through the Golgi (A) Time-lapse spinning disk confocal microscopy was used to image cargo (ss-DsRed) accumulated in the perinuclear region of cells lacking Sar1 following addition of 1,6-hexanediol (top) or 2,5- hexanediol (bottom). Representative images are shown (n = 10 cells; three biological replicates). Scale bar, 5 mm. (B) Quantiﬁcation of cargo (ss-DsRed) remaining within the perinuclear region of cells lacking Sar1 following 30 min of incubation with 1,6-hexanediol or 2,5-hexanediol (relative to the 0 min time point). Error bars represent mean ± SEM (n = 10 cells each; three biological replicates). ***p < 0.001, calculated using a t test, compared with treatment with 2,5-hexanediol. (C) Representative electron micrographs taken within the perinuclear region of high-pressure- frozen cells lacking Sar1a following a mock siRNA treatment or Sar1b depletion. The nuclear enve- lope (NE) is indicated in each image (n = 5 cells each; at least three biological replicates). Scale bar, 500 nm. See also Figure S9.

Techniques: Confocal Microscopy, Incubation

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