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Addgene inc davidson lab mcherry constructs

PA-Rac1-induced lamellipodia formation (A) Schematic representation of subdomains in lamellipodia. (B) COS-7 cells expressing PA-Rac1 and <t>Lifeact-mCherry</t> on glass-bottom dishes. Images were captured every 10 s, with PA-Rac1 activated by a 458 nm laser from time 0 at 10-s intervals. The fluorescence images of Lifeact-mCherry are shown. (C) Lifeact-mCherry image of COS-7 cells, highlighting the crescent-shaped cell edges in red. (D–F) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on EM grids. Images were captured every 10 s from −5 min, with PA-Rac1 activated by scanning with a single 458 nm laser within 0–10 min at 10-s intervals. (D and E) Representative time-lapse images. A merged image of DIC and fluorescence of Lifeact-mCherry (yellow) (D), and fluorescence images of Lifeact-mCherry (E) are shown. A yellow arrowhead indicates dotted actin structures within the interior of cells. (F) Quantification of cell area changes. Data are presented as the means ± SD from 6 cells. Blue background indicates the activation period; red dotted line marks the timing of freezing. Scale bar = 20 μm. See also and and .

Davidson Lab Mcherry Constructs, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics"

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

Journal: iScience

doi: 10.1016/j.isci.2025.112529

PA-Rac1-induced lamellipodia formation (A) Schematic representation of subdomains in lamellipodia. (B) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on glass-bottom dishes. Images were captured every 10 s, with PA-Rac1 activated by a 458 nm laser from time 0 at 10-s intervals. The fluorescence images of Lifeact-mCherry are shown. (C) Lifeact-mCherry image of COS-7 cells, highlighting the crescent-shaped cell edges in red. (D–F) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on EM grids. Images were captured every 10 s from −5 min, with PA-Rac1 activated by scanning with a single 458 nm laser within 0–10 min at 10-s intervals. (D and E) Representative time-lapse images. A merged image of DIC and fluorescence of Lifeact-mCherry (yellow) (D), and fluorescence images of Lifeact-mCherry (E) are shown. A yellow arrowhead indicates dotted actin structures within the interior of cells. (F) Quantification of cell area changes. Data are presented as the means ± SD from 6 cells. Blue background indicates the activation period; red dotted line marks the timing of freezing. Scale bar = 20 μm. See also and and .

Figure Legend Snippet: PA-Rac1-induced lamellipodia formation (A) Schematic representation of subdomains in lamellipodia. (B) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on glass-bottom dishes. Images were captured every 10 s, with PA-Rac1 activated by a 458 nm laser from time 0 at 10-s intervals. The fluorescence images of Lifeact-mCherry are shown. (C) Lifeact-mCherry image of COS-7 cells, highlighting the crescent-shaped cell edges in red. (D–F) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on EM grids. Images were captured every 10 s from −5 min, with PA-Rac1 activated by scanning with a single 458 nm laser within 0–10 min at 10-s intervals. (D and E) Representative time-lapse images. A merged image of DIC and fluorescence of Lifeact-mCherry (yellow) (D), and fluorescence images of Lifeact-mCherry (E) are shown. A yellow arrowhead indicates dotted actin structures within the interior of cells. (F) Quantification of cell area changes. Data are presented as the means ± SD from 6 cells. Blue background indicates the activation period; red dotted line marks the timing of freezing. Scale bar = 20 μm. See also and and .

Techniques Used: Expressing, Fluorescence, Activation Assay

Experimental workflow from sample preparation to tomogram acquisition Laminin-coated EM grids were placed on the 3D-printed grid holders in 4-well dishes, and cells were seeded on the EM grids. At the same time, plasmids encoded mVenus-PA-Rac1 and Lifeact-mCherry were transfected into the cells. The next day, the cells were rapidly frozen using a plunge freezer after inducing lamellipodia formation by activating PA-Rac1 with blue light irradiation, controlling the timing of the freezing. The frozen cells were observed using cryo-fluorescence microscopy to identify the cells that had formed lamellipodia. By correlating the fluorescence images with low-magnification EM images, the regions for tomography were determined. Subsequently, continuous tilt series images were acquired by cryo-EM from −70° to +70°, and tomograms were reconstructed.

Figure Legend Snippet: Experimental workflow from sample preparation to tomogram acquisition Laminin-coated EM grids were placed on the 3D-printed grid holders in 4-well dishes, and cells were seeded on the EM grids. At the same time, plasmids encoded mVenus-PA-Rac1 and Lifeact-mCherry were transfected into the cells. The next day, the cells were rapidly frozen using a plunge freezer after inducing lamellipodia formation by activating PA-Rac1 with blue light irradiation, controlling the timing of the freezing. The frozen cells were observed using cryo-fluorescence microscopy to identify the cells that had formed lamellipodia. By correlating the fluorescence images with low-magnification EM images, the regions for tomography were determined. Subsequently, continuous tilt series images were acquired by cryo-EM from −70° to +70°, and tomograms were reconstructed.

Techniques Used: Sample Prep, Transfection, Irradiation, Fluorescence, Microscopy, Tomography, Cryo-EM Sample Prep

Cryo-CLEM images Cryo-CLEM images of three cells from which tomograms were captured in this study. Green: mVenus-PA-Rac1, purple: Lifeact-mCherry. See also .

Figure Legend Snippet: Cryo-CLEM images Cryo-CLEM images of three cells from which tomograms were captured in this study. Green: mVenus-PA-Rac1, purple: Lifeact-mCherry. See also .

Techniques Used:

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Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: PA-Rac1-induced lamellipodia formation (A) Schematic representation of subdomains in lamellipodia. (B) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on glass-bottom dishes. Images were captured every 10 s, with PA-Rac1 activated by a 458 nm laser from time 0 at 10-s intervals. The fluorescence images of Lifeact-mCherry are shown. (C) Lifeact-mCherry image of COS-7 cells, highlighting the crescent-shaped cell edges in red. (D–F) COS-7 cells expressing PA-Rac1 and Lifeact-mCherry on EM grids. Images were captured every 10 s from −5 min, with PA-Rac1 activated by scanning with a single 458 nm laser within 0–10 min at 10-s intervals. (D and E) Representative time-lapse images. A merged image of DIC and fluorescence of Lifeact-mCherry (yellow) (D), and fluorescence images of Lifeact-mCherry (E) are shown. A yellow arrowhead indicates dotted actin structures within the interior of cells. (F) Quantification of cell area changes. Data are presented as the means ± SD from 6 cells. Blue background indicates the activation period; red dotted line marks the timing of freezing. Scale bar = 20 μm. See also and and .

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques: Expressing, Fluorescence, Activation Assay

Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: Experimental workflow from sample preparation to tomogram acquisition Laminin-coated EM grids were placed on the 3D-printed grid holders in 4-well dishes, and cells were seeded on the EM grids. At the same time, plasmids encoded mVenus-PA-Rac1 and Lifeact-mCherry were transfected into the cells. The next day, the cells were rapidly frozen using a plunge freezer after inducing lamellipodia formation by activating PA-Rac1 with blue light irradiation, controlling the timing of the freezing. The frozen cells were observed using cryo-fluorescence microscopy to identify the cells that had formed lamellipodia. By correlating the fluorescence images with low-magnification EM images, the regions for tomography were determined. Subsequently, continuous tilt series images were acquired by cryo-EM from −70° to +70°, and tomograms were reconstructed.

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques: Sample Prep, Transfection, Irradiation, Fluorescence, Microscopy, Tomography, Cryo-EM Sample Prep

Journal: iScience

Article Title: Cryo-ET of actin cytoskeleton and membrane structure in lamellipodia formation using optogenetics

doi: 10.1016/j.isci.2025.112529

Figure Lengend Snippet: Cryo-CLEM images Cryo-CLEM images of three cells from which tomograms were captured in this study. Green: mVenus-PA-Rac1, purple: Lifeact-mCherry. See also .

Article Snippet: mCherry-ARP2-N-14 , Davidson lab – mCherry constructs, unpublished , RRID:Addgene 54980.

Techniques:

Figure 1. A rapid and efficient pipeline for CRISPR/Cas9 knock-in of fluorescent proteins at the endogenous locus of Rab11 family members. (A–C) Schematic representation of single-stranded DNA (ssDNA) mediated knock-in strategy of different fluorophores with corresponding homologous arms (A), the ssDNA preparation technique (IVT=in vitro transcription; RT=reverse transcription; RE=restriction endonuclease; purif.=purification using magnetic SPRI beads) (B), and our optimized pipeline (RNP=ribonucleoprotein complex) (C). Illustrations were created with BioRender.com. FACS and fluorescence widefield images (background subtracted) of mNeonGreen or mCherry knock-ins to Rab11a or Rab11b loci of (D) A2780 or (E) COV362

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 1. A rapid and efficient pipeline for CRISPR/Cas9 knock-in of fluorescent proteins at the endogenous locus of Rab11 family members. (A–C) Schematic representation of single-stranded DNA (ssDNA) mediated knock-in strategy of different fluorophores with corresponding homologous arms (A), the ssDNA preparation technique (IVT=in vitro transcription; RT=reverse transcription; RE=restriction endonuclease; purif.=purification using magnetic SPRI beads) (B), and our optimized pipeline (RNP=ribonucleoprotein complex) (C). Illustrations were created with BioRender.com. FACS and fluorescence widefield images (background subtracted) of mNeonGreen or mCherry knock-ins to Rab11a or Rab11b loci of (D) A2780 or (E) COV362

Article Snippet: Recombinant DNA reagent pCDH- TRE3GS- EF1a- tagBFPT2A- TetOn3G Andrew Gilmore lab Lentiviral tetracycline inducible backbone Transfected construct (synthetic) pCDH- EF1a- tagBFP- T2ATetOn3G this paper RRID:Addgene_179888 Lentiviral construct to express TetOn3G Transfected construct (synthetic) pCDH- EF1a- HygroR- T2ATetOn3G this paper RRID:Addgene_179887 Lentiviral construct to express TetOn3G Transfected construct (human) mCherry- Rab11a Michael Davidson lab RRID:Addgene_55124 Mammalian Expression vector for transfection Transfected construct (human) GFP- Rab11b Marci Scidmore lab Mammalian Expression vector for transfection Transfected construct (human) GFP- Rab11a DOI:10.1016/j. devcel.2007.08.012 Mammalian Expression vector for transfection Transfected construct (Arabidopsis thaliana) pSH- EFIRES- P- AtAFB2 DOI:10.1038/s41592-019-0512-x RRID:Addgene_129715 Mammalian Expression, CRISPR, and TALEN Transfected construct (A. thaliana) pCDH- AtAFB2- mTagBFP2 this paper RRID:Addgene_179889 Lentiviral construct Transfected construct (synthetic) pMito- mCherry- FRB DOI:10.1242/jcs.124834 RRID:Addgene_59352 Mammalian Expression vector for transfection Transfected construct (synthetic) GFP- FKBP Stephen Royle lab Mammalian Expression vector for transfection Transfected construct (synthetic) pMito- iRFP670- FRB this paper Mammalian Expression vector for transfection Transfected construct (human) pJET- 50- DExogron- mCherryR11b this paper RRID:Addgene_179904 CRISPR and knock- in donor Transfected construct (human) pJET- 49- DExogronmNeonGreen- R11a this paper RRID:Addgene_179903 CRISPR and knock- in donor Transfected construct (human) pJET- 42- DExConantiGFPnanobody- mCherryR11a this paper RRID:Addgene_179902 CRISPR and knock- in donor Continued on next page Continued Gemperle et al. eLife 2022;11:e76651.

Techniques: CRISPR, Knock-In, In Vitro, Reverse Transcription, Purification, Fluorescence

Figure 2. Reversible suppression of endogenous gene expression with DExCon (Doxycycline-mediated endogenous gene Expression Control). (A) DExCon schematic. (B) DExCon knock-in cells were doxycycline (dox) treated for 24–72 hr, sorted for mCherry or mNeonGreen fluorescence, and grown for 2 weeks without dox before re-analysis. Representative immunoblots of mNeonGreen-Rab11a or mCherry-Rab11b DExCon A2780 cells treated or not treated with dox for 48 hr (a=Rab11a; b=Rab11b) probed with anti-mNeonGreen (mNG), anti-mCherry (RFP), anti-Rab11a, or anti-Rab11 (targeting both Rab11a/b) antibodies shown as black and white. Tubulin (Tub), loading control. CTRL represents wt A2780 over-expressing Lifeact-

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 2. Reversible suppression of endogenous gene expression with DExCon (Doxycycline-mediated endogenous gene Expression Control). (A) DExCon schematic. (B) DExCon knock-in cells were doxycycline (dox) treated for 24–72 hr, sorted for mCherry or mNeonGreen fluorescence, and grown for 2 weeks without dox before re-analysis. Representative immunoblots of mNeonGreen-Rab11a or mCherry-Rab11b DExCon A2780 cells treated or not treated with dox for 48 hr (a=Rab11a; b=Rab11b) probed with anti-mNeonGreen (mNG), anti-mCherry (RFP), anti-Rab11a, or anti-Rab11 (targeting both Rab11a/b) antibodies shown as black and white. Tubulin (Tub), loading control. CTRL represents wt A2780 over-expressing Lifeact-

Techniques: Gene Expression, Control, Knock-In, Fluorescence, Western Blot, Expressing

Figure 3. Spatiotemporal control of gene re-activation with DExCon (Doxycycline-mediated endogenous gene Expression Control) and LUXon (light responsive DExCon). (A) Immunoblots of A2780 cells lysates probed with antibodies specific for anti-Rab11a, targeting both Rab11a/b (Rab11) or Rab25 (a=Rab11a; b=Rab11b). Tubulin, loading control. (B) Schematic of the DExCon-mNeonGreen knock-in strategy and lentiviral transduction for doxycycline (dox)-dependent re-activation of Rab25 expression. (C–D) Rab25 DExCon knock-in cells were dox treated for 24–72 hr, sorted for

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 3. Spatiotemporal control of gene re-activation with DExCon (Doxycycline-mediated endogenous gene Expression Control) and LUXon (light responsive DExCon). (A) Immunoblots of A2780 cells lysates probed with antibodies specific for anti-Rab11a, targeting both Rab11a/b (Rab11) or Rab25 (a=Rab11a; b=Rab11b). Tubulin, loading control. (B) Schematic of the DExCon-mNeonGreen knock-in strategy and lentiviral transduction for doxycycline (dox)-dependent re-activation of Rab25 expression. (C–D) Rab25 DExCon knock-in cells were dox treated for 24–72 hr, sorted for

Techniques: Control, Activation Assay, Gene Expression, Western Blot, Knock-In, Transduction, Expressing

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 4. Simultaneous visualization of Rab11 family members. (A) Schematic of triple knock-in A2780 cells (mNeonGreen-Rab11a, mCherry-Rab11b, DExCon mTagBFP2-Rab25) created with BioRender.com. (B–D) mNeonGreen-Rab11a/mCherry-Rab11b knock-in A2708 cells were further modified with a DExCon-mTag-BFP2 module at the Rab25 locus (Tet-On transactivator introduced by lentivirus with hygromycin selection). Airyscan confocal fluorescence images of triple knock-in cells treated by doxycycline (dox) (>94 hr) trafficking Alexa-647 labeled transferrin (TFN-647, 15–60 min). Colors represent Rab11s as indicated and line profile corresponds to yellow dashed line. (B) Scale bar=5µm. (C) Maximum intensity Z-projections: top (2D, FN- coated), bottom (3D cell-derived matrix [CDM]). Scale bar=20µm; see also Videos 9–10. (D–E) dox induced (>94 hr) triple knock-in A2780 cells recycling

Techniques: Knock-In, Modification, Selection, Fluorescence, Labeling, Derivative Assay

Figure 5. DExCon (Doxycycline-mediated endogenous gene Expression Control) reveals protein expression kinetics and dynamics of relocalization. (A) Immunoblots of mNeonGreen-Rab11a and mCherry-Rab11b DExCon or double DExCon A2780 cells (top) and mNeonGreen-Rab25 DExCon cells (bottom) treated or not treated with doxycycline (dox) for the time indicated (hours). Anti-mNeonGreen (mNG), anti-mCherry (RFP; mCH), anti- Rab25, anti-Rab11a, or anti-Rab11 targeting both Rab11a/b (Rab11) were used to probe expression levels. Tubulin (Tub), loading control. M=marker;

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 5. DExCon (Doxycycline-mediated endogenous gene Expression Control) reveals protein expression kinetics and dynamics of relocalization. (A) Immunoblots of mNeonGreen-Rab11a and mCherry-Rab11b DExCon or double DExCon A2780 cells (top) and mNeonGreen-Rab25 DExCon cells (bottom) treated or not treated with doxycycline (dox) for the time indicated (hours). Anti-mNeonGreen (mNG), anti-mCherry (RFP; mCH), anti- Rab25, anti-Rab11a, or anti-Rab11 targeting both Rab11a/b (Rab11) were used to probe expression levels. Tubulin (Tub), loading control. M=marker;

Techniques: Gene Expression, Control, Expressing, Western Blot, Marker

Figure 7. Rab11 expression levels modulate transferrin (TFN) receptor recycling. (A) Schematic illustration (created with Biorender.com) of TFN- 647 recycling assay with depicted pathways: rapid direct route from early endosomes to the plasma membrane and a slower/longer route through perinuclear recycling endosomes via Rab11. (B–C) TFN-647 recycling assay with A2780 DExogron-mCherry-Rab11b, DExCon-mNeonGreen-Rab11a cells or their combination described previously in Figure 6, mNeonGreen-Rab25 DExcon cells sorted for high as shown in Figure 3—figure supplement 1D. Cells doxycycline (dox) pre-treated follow by ±dox/Indole-3-acetic acid (IAA) or their combinations as described; dox (250 ng/ml); IAA (100 in B or 10 μg/ ml in C). TFN-647 recycled levels were measured at 30 min (B) or 10 min (C) by flow cytometry and normalized to TFN-647 internalization levels (reached in 30 min) as % of internal pool. IAA or dox/IAA condition in (C) required extra normalization based on the wt control to subtract the negative effect of IAA. wt=CRISPR unmodified A2780 cells stably expressing AtAFBP2 and Teto3G-T2A-HygroR. FACS analysis of mNeonGreen/mCherry fluorescence levels shown below graphs (B–C), dashed line indicates negative Ctrl. Log10 scale shown. Black rectangles indicate subpopulations (high/low) analyzed for TFN-647 recycling rate. Classical Rab11a/Rab11b double knock-in cells used as reference for mNeonGreen/mCherry brightness. Box and whiskers (geometrical mean) are shown from three independent biological replicates (B–C). One-way ordinary or repeated-measures ANOVA analysis Tukey post hoc test used for statistical analysis and compared to Ctrl (B; 30 min recycling) or among different conditions across modified Rab11a/b/25 toward untreated cells or as indicated (B–C).

Journal: eLife

Article Title: On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members

doi: 10.7554/elife.76651

Figure Lengend Snippet: Figure 7. Rab11 expression levels modulate transferrin (TFN) receptor recycling. (A) Schematic illustration (created with Biorender.com) of TFN- 647 recycling assay with depicted pathways: rapid direct route from early endosomes to the plasma membrane and a slower/longer route through perinuclear recycling endosomes via Rab11. (B–C) TFN-647 recycling assay with A2780 DExogron-mCherry-Rab11b, DExCon-mNeonGreen-Rab11a cells or their combination described previously in Figure 6, mNeonGreen-Rab25 DExcon cells sorted for high as shown in Figure 3—figure supplement 1D. Cells doxycycline (dox) pre-treated follow by ±dox/Indole-3-acetic acid (IAA) or their combinations as described; dox (250 ng/ml); IAA (100 in B or 10 μg/ ml in C). TFN-647 recycled levels were measured at 30 min (B) or 10 min (C) by flow cytometry and normalized to TFN-647 internalization levels (reached in 30 min) as % of internal pool. IAA or dox/IAA condition in (C) required extra normalization based on the wt control to subtract the negative effect of IAA. wt=CRISPR unmodified A2780 cells stably expressing AtAFBP2 and Teto3G-T2A-HygroR. FACS analysis of mNeonGreen/mCherry fluorescence levels shown below graphs (B–C), dashed line indicates negative Ctrl. Log10 scale shown. Black rectangles indicate subpopulations (high/low) analyzed for TFN-647 recycling rate. Classical Rab11a/Rab11b double knock-in cells used as reference for mNeonGreen/mCherry brightness. Box and whiskers (geometrical mean) are shown from three independent biological replicates (B–C). One-way ordinary or repeated-measures ANOVA analysis Tukey post hoc test used for statistical analysis and compared to Ctrl (B; 30 min recycling) or among different conditions across modified Rab11a/b/25 toward untreated cells or as indicated (B–C).

Techniques: Expressing, Clinical Proteomics, Membrane, Flow Cytometry, Control, CRISPR, Stable Transfection, Fluorescence, Knock-In, Modification

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