Construct:Article Title: An Optogenetic approach to control protein localization during embryogenesis of the sea urchin
Article Snippet: All subcloning reactions were designed to be in frame with each gene of interest, and were conducted by the In-Fusion HD Cloning kit (Clonetech) protocol. .. To construct pSP64-GFP, the GFP open reading frame (ORF) was amplified via PCR from pSP64-Vasa-GFP ( Yajima and Wessel, 2011 ) and inserted at the Apa1 site of the vector. pCS2–2x mCherry-EMTB (# 26742) and pCS2-membrane-mCherry (#53750) were obtained from Addgene. pSP64-Vasa-mCherry was constructed by replacing the GFP sequence of pSP64-Vasa-GFP at the Spe1 site. ..
Amplification:Article Title: An Optogenetic approach to control protein localization during embryogenesis of the sea urchin
Article Snippet: All subcloning reactions were designed to be in frame with each gene of interest, and were conducted by the In-Fusion HD Cloning kit (Clonetech) protocol. .. To construct pSP64-GFP, the GFP open reading frame (ORF) was amplified via PCR from pSP64-Vasa-GFP ( Yajima and Wessel, 2011 ) and inserted at the Apa1 site of the vector. pCS2–2x mCherry-EMTB (# 26742) and pCS2-membrane-mCherry (#53750) were obtained from Addgene. pSP64-Vasa-mCherry was constructed by replacing the GFP sequence of pSP64-Vasa-GFP at the Spe1 site. ..
Polymerase Chain Reaction:Article Title: An Optogenetic approach to control protein localization during embryogenesis of the sea urchin
Article Snippet: All subcloning reactions were designed to be in frame with each gene of interest, and were conducted by the In-Fusion HD Cloning kit (Clonetech) protocol. .. To construct pSP64-GFP, the GFP open reading frame (ORF) was amplified via PCR from pSP64-Vasa-GFP ( Yajima and Wessel, 2011 ) and inserted at the Apa1 site of the vector. pCS2–2x mCherry-EMTB (# 26742) and pCS2-membrane-mCherry (#53750) were obtained from Addgene. pSP64-Vasa-mCherry was constructed by replacing the GFP sequence of pSP64-Vasa-GFP at the Spe1 site. ..
Plasmid Preparation:Article Title: An Optogenetic approach to control protein localization during embryogenesis of the sea urchin
Article Snippet: All subcloning reactions were designed to be in frame with each gene of interest, and were conducted by the In-Fusion HD Cloning kit (Clonetech) protocol. .. To construct pSP64-GFP, the GFP open reading frame (ORF) was amplified via PCR from pSP64-Vasa-GFP ( Yajima and Wessel, 2011 ) and inserted at the Apa1 site of the vector. pCS2–2x mCherry-EMTB (# 26742) and pCS2-membrane-mCherry (#53750) were obtained from Addgene. pSP64-Vasa-mCherry was constructed by replacing the GFP sequence of pSP64-Vasa-GFP at the Spe1 site. ..
Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer
Article Snippet: .. The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI). .. The following primary antibodies were used for immunofluorescence and/or Western blots: anti- Wnt3 (Abcam 116222), anti- Flotillin- 2 (Abcam 96507), anti- Flotillin- 2 (Abcam ab113661), anti- Ror2 (Santa Cruz H- 1), anti- beta- actin (Proteintech 60008–1- Ig), anti- Myosin- X (Santa Cruz C- 1), and anti- Evi (EMD Milipore YJ5).
Sequencing:Article Title: An Optogenetic approach to control protein localization during embryogenesis of the sea urchin
Article Snippet: All subcloning reactions were designed to be in frame with each gene of interest, and were conducted by the In-Fusion HD Cloning kit (Clonetech) protocol. .. To construct pSP64-GFP, the GFP open reading frame (ORF) was amplified via PCR from pSP64-Vasa-GFP ( Yajima and Wessel, 2011 ) and inserted at the Apa1 site of the vector. pCS2–2x mCherry-EMTB (# 26742) and pCS2-membrane-mCherry (#53750) were obtained from Addgene. pSP64-Vasa-mCherry was constructed by replacing the GFP sequence of pSP64-Vasa-GFP at the Spe1 site. ..
Transfection:Article Title: WNT7A-positive dendritic cytonemes control synaptogenesis in cortical neurons
Article Snippet: .. The following plasmids were used in transfections: pCS2+-membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene # 14757 ), pcDNA3.1-WNT7A-GFP, pcDNA3.2-WNT7A-V5 (Addgene # 43816 ), 7×TRE-SuperTOPFlash-NLS-mCherry , pCS2+-LifeAct-GFP, pCAG-PSD95.FingR-eGFP-CCR5TC (Addgene # 46295 ), Lck-mScarlet-I (Addgene # 98821 ), pCS2+-Vhh-CD8-mCherry [cloned from Gal4/LexA-Vhh-CD8-mCherry (M. Affolter, University of Basel, Switzerland) into pCS2+ vector using XhoI/XbaI], pCS2+-GAP43-jGCaMP7 s [cloned from pGP-CMV-jGCaMP7 s (Addgene # 104463 ) into pCS2+-GAP43-GFP using XbaI/SnaBI], pCS2+LRP6-eGFP (gift from G. Davidson, KIT, Karlsruhe, Germany), GFP-Bsn 95-3938 (gift from Eckart Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany), pcDNA3.1-Notum-CD8-mCh [assembled by Gibson cloning with pcDNA3.1-hNotumFL (gift from J. P. Vincent, Francis Crick Institute, London, UK) and pCS2+-Vhh-CD8-mCherry], PSD-95-pTagRFP (Addgene # 52671 ). .. The following primary antibodies were used for immunofluorescence: anti-WNT7A (abcam, ab100792), anti-LRP6 (BioTechne, FAB1505R), anti-bassoon (abcam, ab82958), anti-PSD95 (abcam, ab18258), anti-PSD95 (abcam, ab13552), anti-flotillin 2 (Santa Cruz Biotechnology, sc-28320), anti-flotillin 2 (abcam, ab113661), anti-myosin-X (C-1) (Santa Cruz Biotechnology, sc-166720), anti-Evi (EMD Millipore, YJ5).
Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer
Article Snippet: .. The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI). .. The following primary antibodies were used for immunofluorescence and/or Western blots: anti- Wnt3 (Abcam 116222), anti- Flotillin- 2 (Abcam 96507), anti- Flotillin- 2 (Abcam ab113661), anti- Ror2 (Santa Cruz H- 1), anti- beta- actin (Proteintech 60008–1- Ig), anti- Myosin- X (Santa Cruz C- 1), and anti- Evi (EMD Milipore YJ5).
Membrane:Article Title: WNT7A-positive dendritic cytonemes control synaptogenesis in cortical neurons
Article Snippet: .. The following plasmids were used in transfections: pCS2+-membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene # 14757 ), pcDNA3.1-WNT7A-GFP, pcDNA3.2-WNT7A-V5 (Addgene # 43816 ), 7×TRE-SuperTOPFlash-NLS-mCherry , pCS2+-LifeAct-GFP, pCAG-PSD95.FingR-eGFP-CCR5TC (Addgene # 46295 ), Lck-mScarlet-I (Addgene # 98821 ), pCS2+-Vhh-CD8-mCherry [cloned from Gal4/LexA-Vhh-CD8-mCherry (M. Affolter, University of Basel, Switzerland) into pCS2+ vector using XhoI/XbaI], pCS2+-GAP43-jGCaMP7 s [cloned from pGP-CMV-jGCaMP7 s (Addgene # 104463 ) into pCS2+-GAP43-GFP using XbaI/SnaBI], pCS2+LRP6-eGFP (gift from G. Davidson, KIT, Karlsruhe, Germany), GFP-Bsn 95-3938 (gift from Eckart Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany), pcDNA3.1-Notum-CD8-mCh [assembled by Gibson cloning with pcDNA3.1-hNotumFL (gift from J. P. Vincent, Francis Crick Institute, London, UK) and pCS2+-Vhh-CD8-mCherry], PSD-95-pTagRFP (Addgene # 52671 ). .. The following primary antibodies were used for immunofluorescence: anti-WNT7A (abcam, ab100792), anti-LRP6 (BioTechne, FAB1505R), anti-bassoon (abcam, ab82958), anti-PSD95 (abcam, ab18258), anti-PSD95 (abcam, ab13552), anti-flotillin 2 (Santa Cruz Biotechnology, sc-28320), anti-flotillin 2 (abcam, ab113661), anti-myosin-X (C-1) (Santa Cruz Biotechnology, sc-166720), anti-Evi (EMD Millipore, YJ5).
Article Title: The scaffolding protein flot2 promotes cytoneme-based transport of wnt3 in gastric cancer
Article Snippet: .. The following plasmids were used in transfections and/or microinjections: pCS2 + membrane- mCherry (Mattes et al., 2018), pCAG- mGFP membrane- bound GFP (Addgene 14757), pEGFP- N1 Flot2- GFP (Neumann- Giesen et al., 2004), pEGFP- N1 ∆N- Flot2- GFP (Neumann- Giesen et al., 2004), 7×TRE SuperTOPFlash- NLS- mCherry (Moro et al., 2012), JNK KTR- mCherry (Regot et al., 2014), pCS2 + LifeAct GFP, pCS2 + Rab5 GFP (Jim Smith Group), Rab7- eGFP (from Rüdiger Rudolf), pEGFP- N1 LAMP1- mTurq2 (Addgene #98828), IRSp534K- mCherry/GFP (Stanganello et al., 2015), pCS2 + Wnt8a- mCherry (Stanganello et al., 2015), pCS2 + Ror2- mCherry (cloned in with ClaI and XbaI), pEGFP- N3 mRor2-∆CRD- GFP (XhoI- XhoI mRor2 insert taken from pcDNA- mRor2, subcloned into SalI site of pEGFP- N3 vector), pCS2 + Ror2- eBFP2 (cloned by inserting eBFP2 into pCS2 + Ror2 plasmid using XbaI and SnaBI), pCS2 + Wnt3- mCherry (cloned from Addgene plasmid pcDNA3.2- Wnt3 (#35909) into pCS2±mCherry vector using ClaI and XbaI). .. The following primary antibodies were used for immunofluorescence and/or Western blots: anti- Wnt3 (Abcam 116222), anti- Flotillin- 2 (Abcam 96507), anti- Flotillin- 2 (Abcam ab113661), anti- Ror2 (Santa Cruz H- 1), anti- beta- actin (Proteintech 60008–1- Ig), anti- Myosin- X (Santa Cruz C- 1), and anti- Evi (EMD Milipore YJ5).
Clone Assay:Article Title: WNT7A-positive dendritic cytonemes control synaptogenesis in cortical neurons
Article Snippet: .. The following plasmids were used in transfections: pCS2+-membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene # 14757 ), pcDNA3.1-WNT7A-GFP, pcDNA3.2-WNT7A-V5 (Addgene # 43816 ), 7×TRE-SuperTOPFlash-NLS-mCherry , pCS2+-LifeAct-GFP, pCAG-PSD95.FingR-eGFP-CCR5TC (Addgene # 46295 ), Lck-mScarlet-I (Addgene # 98821 ), pCS2+-Vhh-CD8-mCherry [cloned from Gal4/LexA-Vhh-CD8-mCherry (M. Affolter, University of Basel, Switzerland) into pCS2+ vector using XhoI/XbaI], pCS2+-GAP43-jGCaMP7 s [cloned from pGP-CMV-jGCaMP7 s (Addgene # 104463 ) into pCS2+-GAP43-GFP using XbaI/SnaBI], pCS2+LRP6-eGFP (gift from G. Davidson, KIT, Karlsruhe, Germany), GFP-Bsn 95-3938 (gift from Eckart Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany), pcDNA3.1-Notum-CD8-mCh [assembled by Gibson cloning with pcDNA3.1-hNotumFL (gift from J. P. Vincent, Francis Crick Institute, London, UK) and pCS2+-Vhh-CD8-mCherry], PSD-95-pTagRFP (Addgene # 52671 ). .. The following primary antibodies were used for immunofluorescence: anti-WNT7A (abcam, ab100792), anti-LRP6 (BioTechne, FAB1505R), anti-bassoon (abcam, ab82958), anti-PSD95 (abcam, ab18258), anti-PSD95 (abcam, ab13552), anti-flotillin 2 (Santa Cruz Biotechnology, sc-28320), anti-flotillin 2 (abcam, ab113661), anti-myosin-X (C-1) (Santa Cruz Biotechnology, sc-166720), anti-Evi (EMD Millipore, YJ5).
Cloning:Article Title: WNT7A-positive dendritic cytonemes control synaptogenesis in cortical neurons
Article Snippet: .. The following plasmids were used in transfections: pCS2+-membrane-mCherry , pCAG-mGFP membrane-bound GFP (Addgene # 14757 ), pcDNA3.1-WNT7A-GFP, pcDNA3.2-WNT7A-V5 (Addgene # 43816 ), 7×TRE-SuperTOPFlash-NLS-mCherry , pCS2+-LifeAct-GFP, pCAG-PSD95.FingR-eGFP-CCR5TC (Addgene # 46295 ), Lck-mScarlet-I (Addgene # 98821 ), pCS2+-Vhh-CD8-mCherry [cloned from Gal4/LexA-Vhh-CD8-mCherry (M. Affolter, University of Basel, Switzerland) into pCS2+ vector using XhoI/XbaI], pCS2+-GAP43-jGCaMP7 s [cloned from pGP-CMV-jGCaMP7 s (Addgene # 104463 ) into pCS2+-GAP43-GFP using XbaI/SnaBI], pCS2+LRP6-eGFP (gift from G. Davidson, KIT, Karlsruhe, Germany), GFP-Bsn 95-3938 (gift from Eckart Gundelfinger, Leibniz Institute for Neurobiology, Magdeburg, Germany), pcDNA3.1-Notum-CD8-mCh [assembled by Gibson cloning with pcDNA3.1-hNotumFL (gift from J. P. Vincent, Francis Crick Institute, London, UK) and pCS2+-Vhh-CD8-mCherry], PSD-95-pTagRFP (Addgene # 52671 ). .. The following primary antibodies were used for immunofluorescence: anti-WNT7A (abcam, ab100792), anti-LRP6 (BioTechne, FAB1505R), anti-bassoon (abcam, ab82958), anti-PSD95 (abcam, ab18258), anti-PSD95 (abcam, ab13552), anti-flotillin 2 (Santa Cruz Biotechnology, sc-28320), anti-flotillin 2 (abcam, ab113661), anti-myosin-X (C-1) (Santa Cruz Biotechnology, sc-166720), anti-Evi (EMD Millipore, YJ5).
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