Structured Review

TaKaRa human microtubule associated protein eb3
Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) <t>mCerulean3-EB3-7</t> (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.
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Images

1) Product Images from "An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching"

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

Journal: PLoS ONE

doi: 10.1371/journal.pone.0017896

Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) mCerulean3-EB3-7 (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.
Figure Legend Snippet: Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) mCerulean3-EB3-7 (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.

Techniques Used: Fluorescence, Imaging, Microscopy

2) Product Images from "Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging"

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

Journal: BMC Biology

doi: 10.1186/1741-7007-6-13

Fluorescence imaging of mTFP1 fusion constructs . (A)-(K) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mTFP1-α-actinin-19 (human non-muscle); (B) mTFP1-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mTFP1-Cx43-7 (rat α-1 connexin-43); (D) mTFP1-keratin-17 (human cytokeratin 18); (E) mTFP1-endoplasmic reticulum-3 (calreticulin signal sequence (51 nucleotides) and KDEL retention sequence); (F) mTFP1-paxillin-22 (chicken); (G) mTFP1-EB3-7 (human microtubule-associated protein; RP/EB family); (H) mTFP1-lysosomes-20 (rat lysosomal membrane glycoprotein 1); (I) mTFP1-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (J) mTFP1-vimentin-7 (human); (K) mTFP1-zyxin-7 (human). (L)-(T) C-terminal fusion constructs: (L) mTFP1-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (M) mTFP1-lamin B1–10 (human); (N) mTFP1-β-actin-7; (O) mTFP1-clathrin light chain-15 (human); (P) mTFP1-fibrillarin-7 (human); (Q) mTFP1-vinculin-23 (human); (R) mTFP1-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S)mTFP1-β-tubulin-6 (human); (T) mTFP1-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras). The cell line used for expressing mTFP1 fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (A), (G), (K), (N) and Q) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.
Figure Legend Snippet: Fluorescence imaging of mTFP1 fusion constructs . (A)-(K) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mTFP1-α-actinin-19 (human non-muscle); (B) mTFP1-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mTFP1-Cx43-7 (rat α-1 connexin-43); (D) mTFP1-keratin-17 (human cytokeratin 18); (E) mTFP1-endoplasmic reticulum-3 (calreticulin signal sequence (51 nucleotides) and KDEL retention sequence); (F) mTFP1-paxillin-22 (chicken); (G) mTFP1-EB3-7 (human microtubule-associated protein; RP/EB family); (H) mTFP1-lysosomes-20 (rat lysosomal membrane glycoprotein 1); (I) mTFP1-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (J) mTFP1-vimentin-7 (human); (K) mTFP1-zyxin-7 (human). (L)-(T) C-terminal fusion constructs: (L) mTFP1-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (M) mTFP1-lamin B1–10 (human); (N) mTFP1-β-actin-7; (O) mTFP1-clathrin light chain-15 (human); (P) mTFP1-fibrillarin-7 (human); (Q) mTFP1-vinculin-23 (human); (R) mTFP1-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S)mTFP1-β-tubulin-6 (human); (T) mTFP1-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras). The cell line used for expressing mTFP1 fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (A), (G), (K), (N) and Q) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

Techniques Used: Fluorescence, Imaging, Construct, Sequencing, Expressing

Live cell imaging of mWasabi fusion vectors . (A)-(J) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mWasabi-α-actinin-19 (human non-muscle); (B) mWasabi-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mWasabi-Cx26-7 (rat β-2 connexin-26); (D) mWasabi-keratin-17 (human cytokeratin 18); (E) mWasabi-paxillin-22 (chicken); (F) mWasabi-EB3-7 (human microtubule-associated protein; RP/EB family); (G) mWasabi-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (H) mWasabi-vimentin-7 (human); (I)mWasabi-H2B-6 (human); (J) mWasabi-zyxin-7 (human). (K)-(T) C-terminal fusion constructs: (K) mWasabi-lamin B1-10 (human); (L) mWasabi-β-actin-7; (M) mWasabi-β-tubulin-6 (human); (N) mWasabi-clathrin light chain-15 (human); (O) mWasabi-vinculin-23 (human); (P) mWasabi-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras); (Q) mWasabi-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (R) mWasabi-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S) mWasabi-endosomes-15 (human RhoB GTPase with an N-terminal c-Myc epitope tag); (T) mWasabi-annexin (A4)-15 (human). The cell line used for expressing mWasabi fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (E) and (F) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.
Figure Legend Snippet: Live cell imaging of mWasabi fusion vectors . (A)-(J) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mWasabi-α-actinin-19 (human non-muscle); (B) mWasabi-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mWasabi-Cx26-7 (rat β-2 connexin-26); (D) mWasabi-keratin-17 (human cytokeratin 18); (E) mWasabi-paxillin-22 (chicken); (F) mWasabi-EB3-7 (human microtubule-associated protein; RP/EB family); (G) mWasabi-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (H) mWasabi-vimentin-7 (human); (I)mWasabi-H2B-6 (human); (J) mWasabi-zyxin-7 (human). (K)-(T) C-terminal fusion constructs: (K) mWasabi-lamin B1-10 (human); (L) mWasabi-β-actin-7; (M) mWasabi-β-tubulin-6 (human); (N) mWasabi-clathrin light chain-15 (human); (O) mWasabi-vinculin-23 (human); (P) mWasabi-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras); (Q) mWasabi-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (R) mWasabi-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S) mWasabi-endosomes-15 (human RhoB GTPase with an N-terminal c-Myc epitope tag); (T) mWasabi-annexin (A4)-15 (human). The cell line used for expressing mWasabi fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (E) and (F) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

Techniques Used: Live Cell Imaging, Construct, Expressing

Related Articles

Clone Assay:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: To generate fusion vectors, the appropriate cloning vector and an EGFP fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: To generate subcellular localization fusion vectors used for experiments in , the appropriate cloning vector and an mEmerald fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

Transfection:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech). .. DNA for mammalian transfection was prepared by either the Plasmid Midi or Maxi kit (QIAGEN).

Amplification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The FPs were amplified with a 5' primer encoding an AgeI site and a 3' primer encoding either a BspEI (C1) or Not1 (N1) site. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Construct:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: All of the other mTFP1 and mWasabi vectors were constructed using C1 and N1 (Clontech-style) cloning vectors. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: N3 and N1 constructs were generated by PCR using previously described methods . .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

Purification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Sequencing:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech). .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene). .. For FRET efficiency and lifetime microscopy experiments, the plasmid encoding the mCerulean:mVenus fusion was provided by Dr. Steven Vogel (NIH) , and was used to generate the mCerulean3:mVenus and mTurquoise:mVenus fusion proteins described in by substituting the coding sequence for mCerulean with the cDNA for mCerulean3.

Generated:

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: N3 and N1 constructs were generated by PCR using previously described methods . .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

Microscopy:

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene). .. For FRET efficiency and lifetime microscopy experiments, the plasmid encoding the mCerulean:mVenus fusion was provided by Dr. Steven Vogel (NIH) , and was used to generate the mCerulean3:mVenus and mTurquoise:mVenus fusion proteins described in by substituting the coding sequence for mCerulean with the cDNA for mCerulean3.

Expressing:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: Paragraph title: Mammalian expression vectors ... Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: Paragraph title: Mammalian expression vectors ... Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

Polymerase Chain Reaction:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: The purified and digested PCR products were ligated into similarly digested EGFP-C1 and EGFP-N1 cloning vector backbones. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: N3 and N1 constructs were generated by PCR using previously described methods . .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

Plasmid Preparation:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech). .. To prepare mTFP1 and mWasabi C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); human fibrillarin, AgeI and BglII (Evrogen); human vinculin, NheI and EcoRI (Open Biosystems, Huntsville, AL); peroximal targeting signal 1 (PTS1 – peroxisomes), AgeI and BspEI (Clontech); chicken protein tyrosine kinase 2, AgeI and BglII (Clare Waterman-Storer, NIH); human annexin (A4), AgeI and BspEI (Alen Piljic, EMBL, Heidelberg); human RhoB GTPase with an N-terminal c-Myc epitope tag (endosomes), AgeI and BspEI (Clontech); and the 20-amino acid farnesylation signal from c-Ha-Ras, AgeI and BspEI (membrane, Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene). .. To prepare mCerulean3 C-terminal fusions, the following digests were performed: human β-actin, NheI and BglII (Clontech); human α-tubulin, NheI and BglII (Clontech); human light chain clathrin, NheI and BglII (George Patterson, NIH); human lamin B1, NheI and BglII (George Patterson, NIH); mouse MAP4, NheI and BglII (mouse microtubule associated protein 4, nucleotides 1918–3135, Richard Cyr, Penn State University); mouse light chain 9 myosin, NheI and BglII (Patricia Wadsworth, University of Massachusetts); human CDC-42, NheI and BglII (OriGene); PCNA, AgeI and BspEI (David Gilbert, FSU); mouse CAF-1, NheI and BglII (Akash Gunjan, FSU); human fibrillarin, AgeI and BspEI (Dimitry Chudakov, Russian Academy of Sciences); human GTPase Rab5a, NheI and BglII (Vicky Allen, University of Manchester).

Gel Purification:

Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging
Article Snippet: To generate fusion vectors, the appropriate cloning vector and an EGFP fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching
Article Snippet: To generate subcellular localization fusion vectors used for experiments in , the appropriate cloning vector and an mEmerald fusion vector were digested, either sequentially or doubly, with the appropriate enzymes and ligated together after gel purification. .. Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

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    TaKaRa human microtubule associated protein eb3
    Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) <t>mCerulean3-EB3-7</t> (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.
    Human Microtubule Associated Protein Eb3, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) mCerulean3-EB3-7 (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.

    Journal: PLoS ONE

    Article Title: An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

    doi: 10.1371/journal.pone.0017896

    Figure Lengend Snippet: Fluorescence imaging of mCerulean3 fusion vectors. Images were recorded in widefield or laser scanning confocal fluorescence microscopy. (A–M) Fusions to the N-terminus of mCerulean3; for each fusion protein the linker amino acid (aa) length is indicated after the name of the targeted organelle or fusion protein. The origin of the targeting cDNA is indicated in parenthesis. (A) mCerulean3-Cx43-7 (rat); (B) mCerulean3-EB3-7 (human microtubule-associated protein; RP/EB family); (C) mCerulean3-Golgi-7 (N-terminal 81 aa of human β-1,4-galactosyltransferase); (D) mCerulean3-α-actinin (human); (E) mCerulean3-PMP-10 (human peroxisomal membrane protein 2); (F) mCerulean3-c-src-7 (chicken c-src tyrosine kinase); (G) mCerulean3-mitochondria-7 (human cytochrome C oxidase subunit VIII); (H) mCerulean3-zyxin-7 (human); (I) mCerulean3-vimentin-7 (human); (J) mCerulean3-lifeact-7 (N-terminal 17 aa from S. cerevisiae Abp 140); (K) mCerulean3-VE-Cadherin-10 (human vascular epithelial cadherin); (L) mCerulean3-fascin-10 (human fascin); (M) mCerulean3-lysosomes-20 (human lysosomal membrane glycoprotein 1; LAMP-1). (N–Y) Fusions to the C-terminus of mCerulean3. (N) mCerulean3-lamin B1-10 (human); (O) mCerulean-MAP4-10 (mouse microtubule associated protein 4, nucleotides 1918–3135); (P) mCerulean3-lc-myosin-10 (mouse myosin light chain 9); (Q) mCerulean3-CDC42-10 (human cell division cycle 42); (R) mCerulean3-α-tubulin-6 (human); (S) mCerulean3-PCNA-19 (human proliferating cell nuclear antigen); (T) mCerulean3-profilin-10 (mouse profilin); (U) mCerulean3-clathrin light chain-15 (human); (V) mCerulean3-CAF1-10 (mouse chromatin assembly factor 1); (W) mCerulean3-fibrillarin-7 (human fibrillarin); (X) mCerulean3-β-actin-7 (human); (Y) mCerulean-Rab5a-7 (human GTPase Rab5a). (Z1–Z5) mCerulean3-H2B-6 (human) illustrating the various phases of mitosis. (Z1) interphase; (Z2) prophase; (Z3) metaphase; (Z4) anaphase; (Z5) early telophase. Scale bars indicate 10 µm.

    Article Snippet: Thus, to prepare mCerulean3 N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 aa of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human peroxisomal membrane protein 2, NotI and AgeI (peroxisomes; OriGene); c-src, BamHI and NotI (chicken c-src tyrosine kinase, Marilyn Resh, Sloan-Kettering Institute); lifeact, BamHI and NotI (N-terminal 17 aa from S. cerevisiae Abp 140, IDT); VE-cadherin, BamHI and NotI (human vascular epithelial cadherin, Andreea Trache, Texas A & M); fascin, BamHI and NotI (human fascin, OriGene).

    Techniques: Fluorescence, Imaging, Microscopy

    Fluorescence imaging of mTFP1 fusion constructs . (A)-(K) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mTFP1-α-actinin-19 (human non-muscle); (B) mTFP1-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mTFP1-Cx43-7 (rat α-1 connexin-43); (D) mTFP1-keratin-17 (human cytokeratin 18); (E) mTFP1-endoplasmic reticulum-3 (calreticulin signal sequence (51 nucleotides) and KDEL retention sequence); (F) mTFP1-paxillin-22 (chicken); (G) mTFP1-EB3-7 (human microtubule-associated protein; RP/EB family); (H) mTFP1-lysosomes-20 (rat lysosomal membrane glycoprotein 1); (I) mTFP1-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (J) mTFP1-vimentin-7 (human); (K) mTFP1-zyxin-7 (human). (L)-(T) C-terminal fusion constructs: (L) mTFP1-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (M) mTFP1-lamin B1–10 (human); (N) mTFP1-β-actin-7; (O) mTFP1-clathrin light chain-15 (human); (P) mTFP1-fibrillarin-7 (human); (Q) mTFP1-vinculin-23 (human); (R) mTFP1-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S)mTFP1-β-tubulin-6 (human); (T) mTFP1-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras). The cell line used for expressing mTFP1 fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (A), (G), (K), (N) and Q) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

    Journal: BMC Biology

    Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    doi: 10.1186/1741-7007-6-13

    Figure Lengend Snippet: Fluorescence imaging of mTFP1 fusion constructs . (A)-(K) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mTFP1-α-actinin-19 (human non-muscle); (B) mTFP1-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mTFP1-Cx43-7 (rat α-1 connexin-43); (D) mTFP1-keratin-17 (human cytokeratin 18); (E) mTFP1-endoplasmic reticulum-3 (calreticulin signal sequence (51 nucleotides) and KDEL retention sequence); (F) mTFP1-paxillin-22 (chicken); (G) mTFP1-EB3-7 (human microtubule-associated protein; RP/EB family); (H) mTFP1-lysosomes-20 (rat lysosomal membrane glycoprotein 1); (I) mTFP1-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (J) mTFP1-vimentin-7 (human); (K) mTFP1-zyxin-7 (human). (L)-(T) C-terminal fusion constructs: (L) mTFP1-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (M) mTFP1-lamin B1–10 (human); (N) mTFP1-β-actin-7; (O) mTFP1-clathrin light chain-15 (human); (P) mTFP1-fibrillarin-7 (human); (Q) mTFP1-vinculin-23 (human); (R) mTFP1-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S)mTFP1-β-tubulin-6 (human); (T) mTFP1-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras). The cell line used for expressing mTFP1 fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (A), (G), (K), (N) and Q) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

    Article Snippet: Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

    Techniques: Fluorescence, Imaging, Construct, Sequencing, Expressing

    Live cell imaging of mWasabi fusion vectors . (A)-(J) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mWasabi-α-actinin-19 (human non-muscle); (B) mWasabi-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mWasabi-Cx26-7 (rat β-2 connexin-26); (D) mWasabi-keratin-17 (human cytokeratin 18); (E) mWasabi-paxillin-22 (chicken); (F) mWasabi-EB3-7 (human microtubule-associated protein; RP/EB family); (G) mWasabi-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (H) mWasabi-vimentin-7 (human); (I)mWasabi-H2B-6 (human); (J) mWasabi-zyxin-7 (human). (K)-(T) C-terminal fusion constructs: (K) mWasabi-lamin B1-10 (human); (L) mWasabi-β-actin-7; (M) mWasabi-β-tubulin-6 (human); (N) mWasabi-clathrin light chain-15 (human); (O) mWasabi-vinculin-23 (human); (P) mWasabi-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras); (Q) mWasabi-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (R) mWasabi-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S) mWasabi-endosomes-15 (human RhoB GTPase with an N-terminal c-Myc epitope tag); (T) mWasabi-annexin (A4)-15 (human). The cell line used for expressing mWasabi fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (E) and (F) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

    Journal: BMC Biology

    Article Title: Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

    doi: 10.1186/1741-7007-6-13

    Figure Lengend Snippet: Live cell imaging of mWasabi fusion vectors . (A)-(J) N-terminal fusion constructs; for each fusion protein the linker amino acid length is indicated after the name of the targeted organelle or fusion protein: (A) mWasabi-α-actinin-19 (human non-muscle); (B) mWasabi-mitochondria-7 (human cytochrome C oxidase subunit VIII); (C) mWasabi-Cx26-7 (rat β-2 connexin-26); (D) mWasabi-keratin-17 (human cytokeratin 18); (E) mWasabi-paxillin-22 (chicken); (F) mWasabi-EB3-7 (human microtubule-associated protein; RP/EB family); (G) mWasabi-golgi-7 (N-terminal 81 amino acids of human β-1,4-galactosyltransferase); (H) mWasabi-vimentin-7 (human); (I)mWasabi-H2B-6 (human); (J) mWasabi-zyxin-7 (human). (K)-(T) C-terminal fusion constructs: (K) mWasabi-lamin B1-10 (human); (L) mWasabi-β-actin-7; (M) mWasabi-β-tubulin-6 (human); (N) mWasabi-clathrin light chain-15 (human); (O) mWasabi-vinculin-23 (human); (P) mWasabi-farnesyl-5 (20-amino acid farnesylation signal from c-Ha-Ras); (Q) mWasabi-focal adhesion kinase-5 (chicken protein tyrosine kinase 2); (R) mWasabi-peroxisomes-2 (peroximal targeting signal 1; PTS1); (S) mWasabi-endosomes-15 (human RhoB GTPase with an N-terminal c-Myc epitope tag); (T) mWasabi-annexin (A4)-15 (human). The cell line used for expressing mWasabi fusion vectors was gray fox lung fibroblast cells (FoLu) in panels (E) and (F) and human cervical adenocarcinoma cells (HeLa) in the remaining panels. Scale bars represent 10 μm.

    Article Snippet: Thus, to prepare mTFP1 and mWasabi N-terminal fusions, the following digests were performed: human non-muscle α-actinin, EcoRI and NotI (vector source, Tom Keller, FSU); human cytochrome C oxidase subunit VIII, BamHI and NotI (mitochondria, Clontech); human zyxin, BamHI and NotI (Clare Waterman-Storer, NIH); rat α-1 connexin-43 and rat β-2 connexin-26, EcoRI and BamHI (Matthias Falk, Lehigh University); human H2B, BamHI and NotI (George Patterson, NIH); N-terminal 81 amino acids of human β-1,4-galactosyltransferase, BamHI and NotI (Golgi, Clontech); human microtubule-associated protein EB3, BamHI and NotI (Lynne Cassimeris, Lehigh University); human vimentin, BamHI and NotI (Robert Goldman, Northwestern University); human keratin 18, EcoRI and NotI (Open Biosystems, Huntsville, AL); chicken paxillin, EcoRI and NotI (Alan Horwitz, University of Virginia); rat lysosomal membrane glycoprotein 1, AgeI and NheI (George Patterson, NIH); endoplasmic reticulum (calreticulin signal sequence and KDEL retention sequence), AgeI and EcoRI (Clontech).

    Techniques: Live Cell Imaging, Construct, Expressing