rabbit anti-gfp Search Results


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  • 99
    Thermo Fisher rabbit anti green fluorescent protein gfp
    Determination of slit expression in the developing visual system using <t>anti-GFP</t> immunocytochemistry. Horizontal sections of E15.5 retina ( A–F ) and ventral diencephalon ( G–I ) stained with a neuron-specific <t>anti-β-tubulin</t> antibody to label the RGC axons ( A , D , G ) or an anti-GFP antibody ( B , C , E , F , H , I ). Boxed regions in A–C are shown at higher power in D–F . In both slit1 +/− ( B , E , H ) and slit2 +/− ( C , F , I ) embryos, tau–GFP is expressed by RGC axons within the OFL at the inner surface of the retina ( B , C , E , F ) and in the optic nerve ( B , C , E , F ), chiasm ( H , I ), and tract ( H , I ). Expression is also present in specific regions of the diencephalon surrounding the developing optic chiasm ( H , I ) and, in slit2 +/− embryos, the cornea and lens epithelium ( C ). NL, Neuroblastic layer; OC, optic chiasm; RGCL, RGC layer; OT, optic tract. Scale bar: A–C , 400 μm; D–F , 100 μm; G–I , 200 μm.
    Rabbit Anti Green Fluorescent Protein Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit anti gfp
    Knockdown of KIF21A inhibits the axonal transport of <t>NCKX2.</t> A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP <t>(pLL-GFP-shKIF21A</t> or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p
    Rabbit Anti Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1383 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad anti gfp
    Early SMA+ cardiomyocytes contribute to the central ventricular conduction system. ( A , A’ ) Whole-mount X-gal staining of the right and left sides of an E18.5 dissected septum after Cre induction at E7.5. Labeled cells are more abundant on the left side and sparse labeling is observed at the crest of the septum (arrow). ( B – D ) Immunofluorescence on sections of E18.5 hearts from Cx40 <t>GFP/+</t> ::Sma Cre/+ ::R26R lacZ/+ mice after Cre activation at E7.5. SMA-derived cells are detected in the right and left bundle branches (BB) and are positive for Cx40-GFP ( B ) and <t>Tbx5</t> ( B’ ) (arrows). SMA+ cells participate to the AVB, that is also positive for Cx40-GFP ( C ) and Tbx5 ( C’ ), but rarely to the AVN which is negative for Cx40-GFP ( D ) but positive for Tbx5 ( D’ ). ( E , F ) Whole-mount X-gal staining of the right and left side of a P7 dissected septum after Cre induction at E7.5. High magnification of the septum crest ( E’ , F’ ) shows scattered X-gal positive cells in the region of the forming central VCS. ( G , H ) Immunofluorescence on sections of a P7 Sma Cre/+ ::R26R lacZ/+ heart after Cre activation at E7.5 showing co-localization of SMA-derived cells and Contactin-2 (Cntn2) in the AVB ( G ) and left Purkinje fibers ( H ), indicated by arrows.
    Anti Gfp, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Immunology Consultant Laboratory rabbit anti green fluorescent protein anti gfp
    Early SMA+ cardiomyocytes contribute to the central ventricular conduction system. ( A , A’ ) Whole-mount X-gal staining of the right and left sides of an E18.5 dissected septum after Cre induction at E7.5. Labeled cells are more abundant on the left side and sparse labeling is observed at the crest of the septum (arrow). ( B – D ) Immunofluorescence on sections of E18.5 hearts from Cx40 <t>GFP/+</t> ::Sma Cre/+ ::R26R lacZ/+ mice after Cre activation at E7.5. SMA-derived cells are detected in the right and left bundle branches (BB) and are positive for Cx40-GFP ( B ) and <t>Tbx5</t> ( B’ ) (arrows). SMA+ cells participate to the AVB, that is also positive for Cx40-GFP ( C ) and Tbx5 ( C’ ), but rarely to the AVN which is negative for Cx40-GFP ( D ) but positive for Tbx5 ( D’ ). ( E , F ) Whole-mount X-gal staining of the right and left side of a P7 dissected septum after Cre induction at E7.5. High magnification of the septum crest ( E’ , F’ ) shows scattered X-gal positive cells in the region of the forming central VCS. ( G , H ) Immunofluorescence on sections of a P7 Sma Cre/+ ::R26R lacZ/+ heart after Cre activation at E7.5 showing co-localization of SMA-derived cells and Contactin-2 (Cntn2) in the AVB ( G ) and left Purkinje fibers ( H ), indicated by arrows.
    Rabbit Anti Green Fluorescent Protein Anti Gfp, supplied by Immunology Consultant Laboratory, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa rabbit anti green fluorescent protein anti gfp antiserum
    GIT1 association with paxillin LD4 is regulated by PIX binding. (A) Paxillin is immunoprecipitated with C-terminal constructs of GIT1. COS-7 cells were transfected with expression vectors containing Flag-tagged ΔGIT1 constructs depicted in panel C and <t>GFP-paxillin.</t> Western blots (WB) of anti-Flag immunoprecipitates or total lysates (50 μg per lane) were probed by <t>antipaxillin.</t> (The smallest Δ3 GIT1 [residues 646 to 770] is detected in separate gels but not shown here.) (B) Coexpression of PIX potentiates the ability of GIT1 to associate with paxillin. GIT1 or Δ4 and Δ3 represent cotransfection. A significant increase of paxillin (top) associated with Flag-GIT1 immunoprecipitates (IP) (middle) was observed when PIX was also present. Total GFP-paxillin expression is shown at the bottom. (C) Summary of constructs used and their ability to bind paxillin via the conserved C-terminal region (black).
    Rabbit Anti Green Fluorescent Protein Anti Gfp Antiserum, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Cell Signaling Technology Inc rabbit polyclonal anti green fluorescent protein anti gfp
    GIT1 association with paxillin LD4 is regulated by PIX binding. (A) Paxillin is immunoprecipitated with C-terminal constructs of GIT1. COS-7 cells were transfected with expression vectors containing Flag-tagged ΔGIT1 constructs depicted in panel C and <t>GFP-paxillin.</t> Western blots (WB) of anti-Flag immunoprecipitates or total lysates (50 μg per lane) were probed by <t>antipaxillin.</t> (The smallest Δ3 GIT1 [residues 646 to 770] is detected in separate gels but not shown here.) (B) Coexpression of PIX potentiates the ability of GIT1 to associate with paxillin. GIT1 or Δ4 and Δ3 represent cotransfection. A significant increase of paxillin (top) associated with Flag-GIT1 immunoprecipitates (IP) (middle) was observed when PIX was also present. Total GFP-paxillin expression is shown at the bottom. (C) Summary of constructs used and their ability to bind paxillin via the conserved C-terminal region (black).
    Rabbit Polyclonal Anti Green Fluorescent Protein Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Torrey Pines Scientific rabbit anti green fluorescent protein gfp
    Hsc70-mediated TRPV1 current inhibition occurs via suppression of TRPV1 channel incorporation at the cell surface. (a) Western blot analysis of TRPV1 and HA-tagged Hsc70 in heat shock treated HEK cells; note that heat shock does not alter either TRPV1 or Hsc70 protein level. (b) Confocal images of HEK cells transfected with TRPV1-pHluorin or TRPV1-pHluorin + Hsc70 with or without heat shock treatment. Non permeabilized cells were <t>immunostained</t> with a <t>GFP</t> antibody (red) to detect TRPV1 at the cell surface. (c) Quantification of membrane versus total expression of TRPV1-pHluorin for different condition of heat shock treatment. Data are expressed as mean values ± SEM ( n = 18). *** P
    Rabbit Anti Green Fluorescent Protein Gfp, supplied by Torrey Pines Scientific, used in various techniques. Bioz Stars score: 90/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit anti green fluorescent protein gfp
    Arp2/3 participates in the CaM and <t>PKCδ</t> regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with <t>GFP</t> (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.
    Rabbit Anti Green Fluorescent Protein Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 90/100, based on 65 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Torrey Pines Biolabs rabbit anti green fluorescent protein anti gfp polyclonal antiserum
    Arp2/3 participates in the CaM and <t>PKCδ</t> regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with <t>GFP</t> (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.
    Rabbit Anti Green Fluorescent Protein Anti Gfp Polyclonal Antiserum, supplied by Torrey Pines Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rdi research diagnostics rabbit polyclonal anti green fluorescent protein gfp
    Arp2/3 participates in the CaM and <t>PKCδ</t> regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with <t>GFP</t> (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.
    Rabbit Polyclonal Anti Green Fluorescent Protein Gfp, supplied by rdi research diagnostics, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology rabbit polyclonal anti green fluorescent protein gfp
    Arp2/3 participates in the CaM and <t>PKCδ</t> regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with <t>GFP</t> (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.
    Rabbit Polyclonal Anti Green Fluorescent Protein Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore rabbit anti green fluorescent protein gfp
    ELOVL4-mediated biosynthesis of VLC-PUFAs is independent of N-glycosylation but requires retention in the ER. A: Western blot showing increased expression of ΔNG compared with WT (left panel) and comparable levels of Δ3His and ΔLys to WT (right panel) in ARPE19 cells. Blots were reprobed for <t>GFP</t> and <t>β-actin.</t> B: ΔNG showed increased levels of 34:5n3 and 36:5n3 elongation products compared with WT in ARPE19 cells supplemented with 20:5n3, correlating with increased expression levels of the protein. ΔLys and Δ3His did not show any detectable level of the elongated VLC-PUFA products. Data are represented as the mean ± SD (n = 3); significance was determined in comparison to WT; * P
    Rabbit Anti Green Fluorescent Protein Gfp, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Determination of slit expression in the developing visual system using anti-GFP immunocytochemistry. Horizontal sections of E15.5 retina ( A–F ) and ventral diencephalon ( G–I ) stained with a neuron-specific anti-β-tubulin antibody to label the RGC axons ( A , D , G ) or an anti-GFP antibody ( B , C , E , F , H , I ). Boxed regions in A–C are shown at higher power in D–F . In both slit1 +/− ( B , E , H ) and slit2 +/− ( C , F , I ) embryos, tau–GFP is expressed by RGC axons within the OFL at the inner surface of the retina ( B , C , E , F ) and in the optic nerve ( B , C , E , F ), chiasm ( H , I ), and tract ( H , I ). Expression is also present in specific regions of the diencephalon surrounding the developing optic chiasm ( H , I ) and, in slit2 +/− embryos, the cornea and lens epithelium ( C ). NL, Neuroblastic layer; OC, optic chiasm; RGCL, RGC layer; OT, optic tract. Scale bar: A–C , 400 μm; D–F , 100 μm; G–I , 200 μm.

    Journal: The Journal of Neuroscience

    Article Title: Slit Proteins Regulate Distinct Aspects of Retinal Ganglion Cell Axon Guidance within Dorsal and Ventral Retina

    doi: 10.1523/JNEUROSCI.1342-06.2006

    Figure Lengend Snippet: Determination of slit expression in the developing visual system using anti-GFP immunocytochemistry. Horizontal sections of E15.5 retina ( A–F ) and ventral diencephalon ( G–I ) stained with a neuron-specific anti-β-tubulin antibody to label the RGC axons ( A , D , G ) or an anti-GFP antibody ( B , C , E , F , H , I ). Boxed regions in A–C are shown at higher power in D–F . In both slit1 +/− ( B , E , H ) and slit2 +/− ( C , F , I ) embryos, tau–GFP is expressed by RGC axons within the OFL at the inner surface of the retina ( B , C , E , F ) and in the optic nerve ( B , C , E , F ), chiasm ( H , I ), and tract ( H , I ). Expression is also present in specific regions of the diencephalon surrounding the developing optic chiasm ( H , I ) and, in slit2 +/− embryos, the cornea and lens epithelium ( C ). NL, Neuroblastic layer; OC, optic chiasm; RGCL, RGC layer; OT, optic tract. Scale bar: A–C , 400 μm; D–F , 100 μm; G–I , 200 μm.

    Article Snippet: The following antibodies were used: rabbit anti-green fluorescent protein (GFP) (1:500; Invitrogen, Paisley, UK), TUJ1 (anti-neuron-specific-β-tubulin) (1:1000; Cambridge Bioscience, Cambridge, UK), anti-phospho-histone H3 (1:100; Upstate Biotechnology, Lake Placid, NY), 39.4D5 (anti-Islet1; 1:50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), and anti-calretinin (1:10,000; Swant, Bellinzona, Switzerland).

    Techniques: Expressing, Immunocytochemistry, Staining

    Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Knockdown of KIF21A inhibits the axonal transport of NCKX2. A , B , shRNA-mediated depletion of KIF21A. A , KIF21A-targeting shRNA (shKIF21A) was expressed by pLentiLox3.7 plasmids (pLL) encoding EGFP or mRFP (pLL-GFP-shKIF21A or pLL-RFP-shKIF21A). FLAG-KIF21A was cotransfected with shKIF21A or empty pLL vectors into HEK293 cells. shKIF21A completely depleted FLAG-KIF21A, but the empty pLL did not. B , Endogenous KIF21A was remarkably depleted in cultured hippocampal neurons infected with lentivirus encoding shKIF21A but not in those with lentivirus encoding nontargeting shRNA (NT control). Time-dependent knockdown of endogenous KIF21A is shown in the right bar graph. The noninfected control is shown in the leftmost bar and the NT control is in the rightmost bar. In A and B , β-actin was detected as a loading control. C , D , The axonal transport of endogenous NCKX2 (green) in the shKIF21A-transfected ( D ) or untransfected control ( C ) neurons. KIF21A-depleted neuron was identified by red fluorescence of mRFP coexpressed with shRNA (insets on DIC images). Dendrites were identified by MAP2 immunofluorescence (red). Neurites that are MAP2 negative but clearly seen in the DIC images (indicated by arrowheads) were regarded as axons. E , Analysis of ADR of endogenous NCKX2. Dendritic ROIs (left; red dotted polygons) were drawn on the endogenous NCKX2 immunofluorescence image (same as in C ). After nullifying pixels that overlap the binary mask of MAP2-positive neurites (middle) from the endogenous NCKX2 image, the axonal ROI was set on the NCKX2 image (right; red dotted polygon). F , The mean ADR (black bars) and DSR (gray bars) of endogenous NCKX2 estimated from the untransfected control ( n = 8) or KIF21A-depleted neurons ( n = 6). ADR of NCKX2 in the KIF21A-depleted group is significantly lower than that in the control group. ** p

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: shRNA, Cell Culture, Infection, Transfection, Fluorescence, Immunofluorescence

    Axonal transport of NCKX2 is inhibited by dnKIF21A. A , Test for functional competence of NCKX2-GFP. Aa , Overexpressed NCKX2-GFP was localized both in the cytoplasm and the plasma membrane in the HEK293 cell. A line profile of GFP fluorescence along the white line is shown in the inset of Aa . Scale bar, 10 μm. Ab , A whole-cell current at the holding potential of 0 mV was recorded from a HEK293 cell expressing NCKX2-GFP using high-Na + and high-BAPTA internal pipette solution. Reverse-mode NCKX current could be induced by bath application of Ca 2+ plus K + , but not by Ca 2+ plus Cs + or Ca 2+ only. B , Overexpression of NCKX2-GFP alone (left) or coexpression of NCKX2-GFP and dnKIF21A (right) in the hippocampal neurons. GFP fluorescence was inverted into gray color to improve contrast. Scale bar, 100 μm. The longest neurites were indicated by arrowheads. C , D , Coexpression of NCKX2-GFP together with KIF21A ( C ) or dnKIF21A ( D ) in hippocampal neurons. The longest neurite was traced from DsRed fluorescence using NeuronJ, and then fluorescence intensity of NCKX2-GFP was measured along the traced line (rightmost graphs). The traced line is overlaid as a red line on each DsRed image (gray). Scale bar, 100 μm. E , Mean NCKX2-GFP fluorescence profiles of the longest neurites. Ten points smoothing was performed with all data. In the range of 50–250 μm, intensity values for NCKX2-GFP from neurons expressing dnKIF21A (red; n = 11) were significantly lower than those from three different control groups: (1) neurons expressing NCKX2-GFP alone (green; n = 19) at all 43 points, (2) neurons expressing KIF21A (black; n = 10) at 39 points, and (3) neurons expressing dnKIF21B (blue; n = 20) at 36 points (mean ± SEM; p

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Axonal transport of NCKX2 is inhibited by dnKIF21A. A , Test for functional competence of NCKX2-GFP. Aa , Overexpressed NCKX2-GFP was localized both in the cytoplasm and the plasma membrane in the HEK293 cell. A line profile of GFP fluorescence along the white line is shown in the inset of Aa . Scale bar, 10 μm. Ab , A whole-cell current at the holding potential of 0 mV was recorded from a HEK293 cell expressing NCKX2-GFP using high-Na + and high-BAPTA internal pipette solution. Reverse-mode NCKX current could be induced by bath application of Ca 2+ plus K + , but not by Ca 2+ plus Cs + or Ca 2+ only. B , Overexpression of NCKX2-GFP alone (left) or coexpression of NCKX2-GFP and dnKIF21A (right) in the hippocampal neurons. GFP fluorescence was inverted into gray color to improve contrast. Scale bar, 100 μm. The longest neurites were indicated by arrowheads. C , D , Coexpression of NCKX2-GFP together with KIF21A ( C ) or dnKIF21A ( D ) in hippocampal neurons. The longest neurite was traced from DsRed fluorescence using NeuronJ, and then fluorescence intensity of NCKX2-GFP was measured along the traced line (rightmost graphs). The traced line is overlaid as a red line on each DsRed image (gray). Scale bar, 100 μm. E , Mean NCKX2-GFP fluorescence profiles of the longest neurites. Ten points smoothing was performed with all data. In the range of 50–250 μm, intensity values for NCKX2-GFP from neurons expressing dnKIF21A (red; n = 11) were significantly lower than those from three different control groups: (1) neurons expressing NCKX2-GFP alone (green; n = 19) at all 43 points, (2) neurons expressing KIF21A (black; n = 10) at 39 points, and (3) neurons expressing dnKIF21B (blue; n = 20) at 36 points (mean ± SEM; p

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Functional Assay, Fluorescence, Expressing, Transferring, Over Expression

    Colocalization of NCKX2 and KIF21A. A , Reliability test of NCKX2 ext antibody. Aa , The HEK293 cells expressing NCKX2-GFP (green) were specifically labeled with anti-NCKX2 ext antibody (red). Untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. Ab , Because the anti-NCKX2 ext antibody was raised against a synthetic peptide within the N-terminal extracellular region (residues 90–102), HEK293 cells expressing NCKX2-FLAG, in which the FLAG tag ( 90 DYKDDDDK 97 ) replaces the N-terminal 8 amino acids of the epitope, were not immunoreactive to the anti-NCKX2 ext (green). Expression of NCKX2-FLAG was detected by anti-FLAG (red), and untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. B , Preabsorption test of anti-NCKX2 ext antibody. DIV18 hippocampal neuron lysate was immunoblotted using anti-NCKX2 ext antibody without (left lane) or with (right lane) preincubation of the antigen peptide. β-Actin was detected as a loading control (bottom panel). C , Endogenous NCKX2 (green) and transfected FLAG-KIF21A (red) were colocalized not only in the somatodendritic region but also in the axon terminals of the cultured hippocampal neuron. Scale bar, 50 μm. D , Higher magnification images of the axonal bouton (dotted box in C ), the soma of which was marked by an arrow in C . The axon was identified by NF-H immunofluorescence (blue). Scale bar, 20 μm. E–G , Quantitative analysis of colocalization. Ea , Higher magnification image of the FLAG-positive axon (boxed region in C ). Other FLAG-negative neurites were eliminated using the binary mask made from NF-H fluorescence image. Eb , The PDM value was calculated at each pixel and shown as the pixel value at its location. For clarity, the contrast of this PDM image was adjusted where the brightness is saturated at 0.2. Scale bar, 50 μm. F , A scattered plot of green and red pixel intensities against their PDM value at each paired pixel. G , The line profiles of NCKX2 (green) and FLAG-KIF21A (red) fluorescence intensities along two ROI lines (red lines in the left panel). The locations of arrowheads in the right two panels correspond to puncta indicated by arrowheads in Eb . *PDM > 0.1; **PDM > 0.2.

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Colocalization of NCKX2 and KIF21A. A , Reliability test of NCKX2 ext antibody. Aa , The HEK293 cells expressing NCKX2-GFP (green) were specifically labeled with anti-NCKX2 ext antibody (red). Untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. Ab , Because the anti-NCKX2 ext antibody was raised against a synthetic peptide within the N-terminal extracellular region (residues 90–102), HEK293 cells expressing NCKX2-FLAG, in which the FLAG tag ( 90 DYKDDDDK 97 ) replaces the N-terminal 8 amino acids of the epitope, were not immunoreactive to the anti-NCKX2 ext (green). Expression of NCKX2-FLAG was detected by anti-FLAG (red), and untransfected cells were visualized by DAPI staining (blue). Scale bar, 20 μm. B , Preabsorption test of anti-NCKX2 ext antibody. DIV18 hippocampal neuron lysate was immunoblotted using anti-NCKX2 ext antibody without (left lane) or with (right lane) preincubation of the antigen peptide. β-Actin was detected as a loading control (bottom panel). C , Endogenous NCKX2 (green) and transfected FLAG-KIF21A (red) were colocalized not only in the somatodendritic region but also in the axon terminals of the cultured hippocampal neuron. Scale bar, 50 μm. D , Higher magnification images of the axonal bouton (dotted box in C ), the soma of which was marked by an arrow in C . The axon was identified by NF-H immunofluorescence (blue). Scale bar, 20 μm. E–G , Quantitative analysis of colocalization. Ea , Higher magnification image of the FLAG-positive axon (boxed region in C ). Other FLAG-negative neurites were eliminated using the binary mask made from NF-H fluorescence image. Eb , The PDM value was calculated at each pixel and shown as the pixel value at its location. For clarity, the contrast of this PDM image was adjusted where the brightness is saturated at 0.2. Scale bar, 50 μm. F , A scattered plot of green and red pixel intensities against their PDM value at each paired pixel. G , The line profiles of NCKX2 (green) and FLAG-KIF21A (red) fluorescence intensities along two ROI lines (red lines in the left panel). The locations of arrowheads in the right two panels correspond to puncta indicated by arrowheads in Eb . *PDM > 0.1; **PDM > 0.2.

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Expressing, Labeling, Staining, FLAG-tag, Transfection, Cell Culture, Immunofluorescence, Fluorescence

    Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.

    Journal: The Journal of Neuroscience

    Article Title: KIF21A-Mediated Axonal Transport and Selective Endocytosis Underlie the Polarized Targeting of NCKX2

    doi: 10.1523/JNEUROSCI.6331-11.2012

    Figure Lengend Snippet: Immunocytochemical localization of endogenous ( A ) or exogenous ( B ) NCKX2 in cultured hippocampal neurons. Aa , DIV23 hippocampal neurons were stained for endogenous NCKX2 (green) and dendritic marker MAP2 (red). Ab–Ae , Surface NCKX2 (s-NCKX2) was immunolabeled by incubating live cells with anti-NCKX2 ext (green) at 36°C, and then cells were fixed, permeabilized, and immunostained with antibodies against axonal marker Tau-1 (red; Ab , Ac ) or presynaptic marker synaptophysin (red; Ad ) or dendritic maker MAP2 (red; Ae ). Higher magnification image of the dashed box in Ab is shown in Ac . The sites where endogenous s-NCKX2 was colocalized with Tau-1 ( Ac ) or synaptophysin ( Ad ) are marked with arrows. Af , Endogenous s-NCKX2 (green) was immunolabeled by the same manner as in Ae except incubating live cells with anti-NCKX2 ext at 4°C before fixation. Scale bars: Aa , 10 μm; Ab , Ae , Af , 50 μm; Ac , Ad , 5 μm. Ba , A DIV7 hippocampal neuron transfected with NCKX2-GFP and DsRed. Scale bar, 50 μm. Bb , A DIV13 hippocampal neuron transfected with NCKX2-GFP (green). Surface NCKX2-GFP was visualized by live-cell immunolabeling with antibody against GFP (s-NCKX2-GFP; red), and then stained for MAP2 (blue). Scale bar, 10 μm. The open and solid arrowheads indicate dendrites and axons, respectively. The asterisks show location of somata.

    Article Snippet: For surface immunostaining of NCKX2, live cells were incubated with rabbit anti-NCKX2ext (1:100) or rabbit anti-GFP (1:100; Millipore Bioscience Research Reagents) in serum-free culture medium for 15 min at 36°C or at 4°C, rinsed with culture medium, fixed with ice-cold 4% PFA or 3.8% formaldehyde in PBS for 20 min, and washed with PBS.

    Techniques: Cell Culture, Staining, Marker, Immunolabeling, Transfection

    Early SMA+ cardiomyocytes contribute to the central ventricular conduction system. ( A , A’ ) Whole-mount X-gal staining of the right and left sides of an E18.5 dissected septum after Cre induction at E7.5. Labeled cells are more abundant on the left side and sparse labeling is observed at the crest of the septum (arrow). ( B – D ) Immunofluorescence on sections of E18.5 hearts from Cx40 GFP/+ ::Sma Cre/+ ::R26R lacZ/+ mice after Cre activation at E7.5. SMA-derived cells are detected in the right and left bundle branches (BB) and are positive for Cx40-GFP ( B ) and Tbx5 ( B’ ) (arrows). SMA+ cells participate to the AVB, that is also positive for Cx40-GFP ( C ) and Tbx5 ( C’ ), but rarely to the AVN which is negative for Cx40-GFP ( D ) but positive for Tbx5 ( D’ ). ( E , F ) Whole-mount X-gal staining of the right and left side of a P7 dissected septum after Cre induction at E7.5. High magnification of the septum crest ( E’ , F’ ) shows scattered X-gal positive cells in the region of the forming central VCS. ( G , H ) Immunofluorescence on sections of a P7 Sma Cre/+ ::R26R lacZ/+ heart after Cre activation at E7.5 showing co-localization of SMA-derived cells and Contactin-2 (Cntn2) in the AVB ( G ) and left Purkinje fibers ( H ), indicated by arrows.

    Journal: Journal of Cardiovascular Development and Disease

    Article Title: Segregation of Central Ventricular Conduction System Lineages in Early SMA+ Cardiomyocytes Occurs Prior to Heart Tube Formation

    doi: 10.3390/jcdd3010002

    Figure Lengend Snippet: Early SMA+ cardiomyocytes contribute to the central ventricular conduction system. ( A , A’ ) Whole-mount X-gal staining of the right and left sides of an E18.5 dissected septum after Cre induction at E7.5. Labeled cells are more abundant on the left side and sparse labeling is observed at the crest of the septum (arrow). ( B – D ) Immunofluorescence on sections of E18.5 hearts from Cx40 GFP/+ ::Sma Cre/+ ::R26R lacZ/+ mice after Cre activation at E7.5. SMA-derived cells are detected in the right and left bundle branches (BB) and are positive for Cx40-GFP ( B ) and Tbx5 ( B’ ) (arrows). SMA+ cells participate to the AVB, that is also positive for Cx40-GFP ( C ) and Tbx5 ( C’ ), but rarely to the AVN which is negative for Cx40-GFP ( D ) but positive for Tbx5 ( D’ ). ( E , F ) Whole-mount X-gal staining of the right and left side of a P7 dissected septum after Cre induction at E7.5. High magnification of the septum crest ( E’ , F’ ) shows scattered X-gal positive cells in the region of the forming central VCS. ( G , H ) Immunofluorescence on sections of a P7 Sma Cre/+ ::R26R lacZ/+ heart after Cre activation at E7.5 showing co-localization of SMA-derived cells and Contactin-2 (Cntn2) in the AVB ( G ) and left Purkinje fibers ( H ), indicated by arrows.

    Article Snippet: Antibodies and Immunofluorescence Antibodies used in this study are specific to Nkx2-5 (Sc8697 Santa-Cruz, Dallas, TX, USA), α-smooth muscle actin (Sigma, F3777), rabbit anti-β-galactosidase (Cappel, MP Biomedicals, Aurora, OH, USA), GFP (AbD Serotec, Bio-Rad, Hercules, CA, USA), Tbx5 (Sc17866 Santa Cruz, Dallas, TX, USA), Hcn4 (AB5808 Millipore, Darmstadt, Germany), and Contactin-2 (AF1714 R & D Systems, Minneapolis, MN, USA).

    Techniques: Staining, Labeling, Immunofluorescence, Mouse Assay, Activation Assay, Derivative Assay

    Localization of PEX10 and PEX12 GFP fusion proteins expressed in T. brucei

    Journal:

    Article Title: Characterization of Glycosomal RING Finger Proteins of Trypanosomatids

    doi: 10.1016/j.exppara.2006.11.004

    Figure Lengend Snippet: Localization of PEX10 and PEX12 GFP fusion proteins expressed in T. brucei

    Article Snippet: Immunoblots were performed as described ( ) using rabbit anti- T. brucei phosphoglycerate kinase ( ) or rabbit anti-GFP antisera, followed by protein A coupled to horseradish peroxidase (Bio-Rad).

    Techniques:

    Interaction between Zwint-1 and ZW10 controls ZW10 kinetochore localization. (A) Immunoblot of Zwint-1 immunoprecipitates shows weak interaction with ZW10. Cells of clone LINT2.8 were subjected to immunoprecipitation with control antibody (Con) or anti-GFP antibody to precipitate Zwint-1 LAPtag and the precipitate was analyzed for the presence of endogenous ZW10. HSS, high speed supernatant before the immunoprecipitation. Bands labeled with asterisks are background due to precipitation from HSS with the anti-GFP antibody. White line indicates that intervening lanes have been spliced out. (B) Analysis of Zwint-1 knockdown efficiency by immunoblot using cells expressing Zwint-1 LAPtag . Lysates of LINT2.8 cells untransfected or transfected with mock or Zwint-1 siRNA plasmid for 72 h were analyzed for Zwint-1 LAPtag (anti-GFP), ZW10, and tubulin expression. Percentage of remaining protein was determined by serial dilution immunoblotting. Band labeled with asterisks is protein that cross reacts with anti-GFP in the LINT2.8 cell line. (C) Immunolocalization of ZW10 in cells depleted of endogenous Zwint-1. HeLa cells transfected as in B were treated with nocodazole for 30 min before fixation and stained for endogenous Zwint-1 and ZW10, and for centromeres (ACA) and DNA (DAPI).

    Journal: The Journal of Cell Biology

    Article Title: ZW10 links mitotic checkpoint signaling to the structural kinetochore

    doi: 10.1083/jcb.200411118

    Figure Lengend Snippet: Interaction between Zwint-1 and ZW10 controls ZW10 kinetochore localization. (A) Immunoblot of Zwint-1 immunoprecipitates shows weak interaction with ZW10. Cells of clone LINT2.8 were subjected to immunoprecipitation with control antibody (Con) or anti-GFP antibody to precipitate Zwint-1 LAPtag and the precipitate was analyzed for the presence of endogenous ZW10. HSS, high speed supernatant before the immunoprecipitation. Bands labeled with asterisks are background due to precipitation from HSS with the anti-GFP antibody. White line indicates that intervening lanes have been spliced out. (B) Analysis of Zwint-1 knockdown efficiency by immunoblot using cells expressing Zwint-1 LAPtag . Lysates of LINT2.8 cells untransfected or transfected with mock or Zwint-1 siRNA plasmid for 72 h were analyzed for Zwint-1 LAPtag (anti-GFP), ZW10, and tubulin expression. Percentage of remaining protein was determined by serial dilution immunoblotting. Band labeled with asterisks is protein that cross reacts with anti-GFP in the LINT2.8 cell line. (C) Immunolocalization of ZW10 in cells depleted of endogenous Zwint-1. HeLa cells transfected as in B were treated with nocodazole for 30 min before fixation and stained for endogenous Zwint-1 and ZW10, and for centromeres (ACA) and DNA (DAPI).

    Article Snippet: Immunoprecipitation and immunoblotting Immunoprecipitation of Zwint-1LAPtag was done as described for the affinity purification, using rabbit anti-GFP antibody coupled and cross-linked to Affi-Prep protein A support (Bio-Rad Laboratories).

    Techniques: Immunoprecipitation, Labeling, Expressing, Transfection, Plasmid Preparation, Serial Dilution, Staining

    GIT1 association with paxillin LD4 is regulated by PIX binding. (A) Paxillin is immunoprecipitated with C-terminal constructs of GIT1. COS-7 cells were transfected with expression vectors containing Flag-tagged ΔGIT1 constructs depicted in panel C and GFP-paxillin. Western blots (WB) of anti-Flag immunoprecipitates or total lysates (50 μg per lane) were probed by antipaxillin. (The smallest Δ3 GIT1 [residues 646 to 770] is detected in separate gels but not shown here.) (B) Coexpression of PIX potentiates the ability of GIT1 to associate with paxillin. GIT1 or Δ4 and Δ3 represent cotransfection. A significant increase of paxillin (top) associated with Flag-GIT1 immunoprecipitates (IP) (middle) was observed when PIX was also present. Total GFP-paxillin expression is shown at the bottom. (C) Summary of constructs used and their ability to bind paxillin via the conserved C-terminal region (black).

    Journal: Molecular and Cellular Biology

    Article Title: Coupling of PAK-Interacting Exchange Factor PIX to GIT1 Promotes Focal Complex Disassembly

    doi:

    Figure Lengend Snippet: GIT1 association with paxillin LD4 is regulated by PIX binding. (A) Paxillin is immunoprecipitated with C-terminal constructs of GIT1. COS-7 cells were transfected with expression vectors containing Flag-tagged ΔGIT1 constructs depicted in panel C and GFP-paxillin. Western blots (WB) of anti-Flag immunoprecipitates or total lysates (50 μg per lane) were probed by antipaxillin. (The smallest Δ3 GIT1 [residues 646 to 770] is detected in separate gels but not shown here.) (B) Coexpression of PIX potentiates the ability of GIT1 to associate with paxillin. GIT1 or Δ4 and Δ3 represent cotransfection. A significant increase of paxillin (top) associated with Flag-GIT1 immunoprecipitates (IP) (middle) was observed when PIX was also present. Total GFP-paxillin expression is shown at the bottom. (C) Summary of constructs used and their ability to bind paxillin via the conserved C-terminal region (black).

    Article Snippet: Anti-HA MAb was from Roche, antipaxillin was from Transduction Laboratories, rabbit anti-green fluorescent protein (GFP) from Clontech, and antiphosphotyrosine (MAb 4G10) was from Upstate Biotechnology.

    Techniques: Binding Assay, Immunoprecipitation, Construct, Transfection, Expressing, Western Blot, Cotransfection

    Hsc70-mediated TRPV1 current inhibition occurs via suppression of TRPV1 channel incorporation at the cell surface. (a) Western blot analysis of TRPV1 and HA-tagged Hsc70 in heat shock treated HEK cells; note that heat shock does not alter either TRPV1 or Hsc70 protein level. (b) Confocal images of HEK cells transfected with TRPV1-pHluorin or TRPV1-pHluorin + Hsc70 with or without heat shock treatment. Non permeabilized cells were immunostained with a GFP antibody (red) to detect TRPV1 at the cell surface. (c) Quantification of membrane versus total expression of TRPV1-pHluorin for different condition of heat shock treatment. Data are expressed as mean values ± SEM ( n = 18). *** P

    Journal: Molecular Pain

    Article Title: The stress protein heat shock cognate 70 (Hsc70) inhibits the Transient Receptor Potential Vanilloid type 1 (TRPV1) channel

    doi: 10.1177/1744806916663945

    Figure Lengend Snippet: Hsc70-mediated TRPV1 current inhibition occurs via suppression of TRPV1 channel incorporation at the cell surface. (a) Western blot analysis of TRPV1 and HA-tagged Hsc70 in heat shock treated HEK cells; note that heat shock does not alter either TRPV1 or Hsc70 protein level. (b) Confocal images of HEK cells transfected with TRPV1-pHluorin or TRPV1-pHluorin + Hsc70 with or without heat shock treatment. Non permeabilized cells were immunostained with a GFP antibody (red) to detect TRPV1 at the cell surface. (c) Quantification of membrane versus total expression of TRPV1-pHluorin for different condition of heat shock treatment. Data are expressed as mean values ± SEM ( n = 18). *** P

    Article Snippet: Immunostaining and confocal microscopy Transfected HEK cells were exposed or not to heat shock (42℃) for 1 h. After fixation in 4% PFA for 5 min, cells were incubated in blocking solution (phosphate-buffered saline, PBS + 1%BSA) for 30 min and then immunostained for 1 h at room temperture with a rabbit anti-green fluorescent protein (GFP) (Torrey Pines Scientific, Carlsbad, CA 92010).

    Techniques: Inhibition, Western Blot, Transfection, Expressing

    Arp2/3 participates in the CaM and PKCδ regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with GFP (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Protein Kinase C? and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    doi: 10.1091/mbc.E07-05-0411

    Figure Lengend Snippet: Arp2/3 participates in the CaM and PKCδ regulated actin dynamics. (A) NRK cells were preincubated with W13 (5 μg/ml; 30 min) and treated with rottlerin (5 μM) and transferrin (50 μg/ml) for 30 min at 37°C. p16, a subunit of the Arp2/3 complex (Alexa Fluor 488) and transferrin-TRITC labeling are shown. (B) Twenty-four hours after transfection with the WA domain of N-WASP tagged with GFP (which binds and sequesters Arp2/3), COS1 cells were incubated with W13 (5 μg/ml; 90 min) and EGF (100 ng/ml; 15 min) at 37°C. Next, cells were fixed and stained with anti-EGFR (Alexa Fluor 594). Bar, 10 μm.

    Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-EGFR (American Type Culture Collection, Manassas, VA), mouse monoclonal anti-actin (Valeant Pharmaceuticals, Costa Mesa, CA), rabbit anti-RhoB (Bethyl Laboratories, Montgomery, TX), mouse monoclonal anti-early endosomal antibody (EEA)1 (BD Biosciences Transduction Laboratories, Erembodegem, Belgium), mouse monoclonal anti-p16-Arc (Synaptic Systems, Göttingen, Germany), rabbit anti-WASP family Verprolin-homologous protein (WAVE) and mouse monoclonal anti-cortactin (Upstate Biotechnology, Charlottesville, VA), rabbit anti-green fluorescent protein (GFP) (Abcam, Cambridge, United Kingdom), rabbit anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Chick Chorioallantoic Membrane Assay, Labeling, Transfection, Incubation, Staining

    Cortactin regulates recycling in the early endosomes. (A) NRK cells were preincubated for 30 min with W13 (5 μg/ml), treated with rottlerin (5 μM) for 30 min, and then with transferrin (50 μg/ml) for 15 min at 37°C. Cortactin (Alexa Fluor 488) and transferrin-TRITC staining are shown. Bar, 10 μm. (B) HeLa cells were transfected for 72 h with cortactin siRNA duplex 1 and 2 or GFP siRNA. Then, cells internalized 125 I-EGF (5 ng/ml; 7 min), and they were treated with W13 (7.5 μg/ml) for 30 min at 37°C to measure recycling and degradation. Each data point in the histogram represents the mean of a minimum of six replicates from two independent experiments for each siRNA (±SD). *p

    Journal: Molecular Biology of the Cell

    Article Title: Protein Kinase C? and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    doi: 10.1091/mbc.E07-05-0411

    Figure Lengend Snippet: Cortactin regulates recycling in the early endosomes. (A) NRK cells were preincubated for 30 min with W13 (5 μg/ml), treated with rottlerin (5 μM) for 30 min, and then with transferrin (50 μg/ml) for 15 min at 37°C. Cortactin (Alexa Fluor 488) and transferrin-TRITC staining are shown. Bar, 10 μm. (B) HeLa cells were transfected for 72 h with cortactin siRNA duplex 1 and 2 or GFP siRNA. Then, cells internalized 125 I-EGF (5 ng/ml; 7 min), and they were treated with W13 (7.5 μg/ml) for 30 min at 37°C to measure recycling and degradation. Each data point in the histogram represents the mean of a minimum of six replicates from two independent experiments for each siRNA (±SD). *p

    Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-EGFR (American Type Culture Collection, Manassas, VA), mouse monoclonal anti-actin (Valeant Pharmaceuticals, Costa Mesa, CA), rabbit anti-RhoB (Bethyl Laboratories, Montgomery, TX), mouse monoclonal anti-early endosomal antibody (EEA)1 (BD Biosciences Transduction Laboratories, Erembodegem, Belgium), mouse monoclonal anti-p16-Arc (Synaptic Systems, Göttingen, Germany), rabbit anti-WASP family Verprolin-homologous protein (WAVE) and mouse monoclonal anti-cortactin (Upstate Biotechnology, Charlottesville, VA), rabbit anti-green fluorescent protein (GFP) (Abcam, Cambridge, United Kingdom), rabbit anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Staining, Transfection

    CaM and PKCδ interact with cortactin. (A) Cellular lysates from HeLa cells were incubated with CaM-Sepharose in the presence of Ca 2+ or EGTA as described in Materials and Methods . All bound fraction and 25 μl of the unbound fraction or lysate was loaded and the presence of cortactin and RhoB was analyzed by Western blotting. (B) HeLa cells were cotransfected with cortactin and GFP or GFP-PKCδ. After 24-h transfection, cells were incubated for 30 min with or without W13 (10 μg/ml) at 37°C. HeLa cell extracts were incubated with anti-GFP polyclonal antibodies, and the immunocomplex was pulled down by using protein A-Sepharose. The presence of cortactin and GFP or GFP-PKCδ in the immunoprecipitates was analyzed by Western blotting by using anti-cortactin and anti-GFP antibodies, respectively. (C) HeLa cells were treated for 30 min ± W13 (10 μg/ml) at 37°C. Cell extracts were incubated with anti-cortactin monoclonal antibody and pulled down using protein G-Sepharose. The presence of PKCδ and cortactin in the immunoprecipitates was analyzed by Western blotting.

    Journal: Molecular Biology of the Cell

    Article Title: Protein Kinase C? and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    doi: 10.1091/mbc.E07-05-0411

    Figure Lengend Snippet: CaM and PKCδ interact with cortactin. (A) Cellular lysates from HeLa cells were incubated with CaM-Sepharose in the presence of Ca 2+ or EGTA as described in Materials and Methods . All bound fraction and 25 μl of the unbound fraction or lysate was loaded and the presence of cortactin and RhoB was analyzed by Western blotting. (B) HeLa cells were cotransfected with cortactin and GFP or GFP-PKCδ. After 24-h transfection, cells were incubated for 30 min with or without W13 (10 μg/ml) at 37°C. HeLa cell extracts were incubated with anti-GFP polyclonal antibodies, and the immunocomplex was pulled down by using protein A-Sepharose. The presence of cortactin and GFP or GFP-PKCδ in the immunoprecipitates was analyzed by Western blotting by using anti-cortactin and anti-GFP antibodies, respectively. (C) HeLa cells were treated for 30 min ± W13 (10 μg/ml) at 37°C. Cell extracts were incubated with anti-cortactin monoclonal antibody and pulled down using protein G-Sepharose. The presence of PKCδ and cortactin in the immunoprecipitates was analyzed by Western blotting.

    Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-EGFR (American Type Culture Collection, Manassas, VA), mouse monoclonal anti-actin (Valeant Pharmaceuticals, Costa Mesa, CA), rabbit anti-RhoB (Bethyl Laboratories, Montgomery, TX), mouse monoclonal anti-early endosomal antibody (EEA)1 (BD Biosciences Transduction Laboratories, Erembodegem, Belgium), mouse monoclonal anti-p16-Arc (Synaptic Systems, Göttingen, Germany), rabbit anti-WASP family Verprolin-homologous protein (WAVE) and mouse monoclonal anti-cortactin (Upstate Biotechnology, Charlottesville, VA), rabbit anti-green fluorescent protein (GFP) (Abcam, Cambridge, United Kingdom), rabbit anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Chick Chorioallantoic Membrane Assay, Incubation, Western Blot, Transfection

    N-WASP or WAVE are not involved in W13 effect. (A) COS1 cells transiently expressing GFP-N-WASP-ΔWA were incubated with W13 (5 μg/ml) for 90 min and EGF (100 ng/ml) for the last 15 min at 37°C, fixed, and immunolabeled with anti-EGFR (Alexa Fluor 594). Bar, 10 μm. (B) NRK cells were preincubated with W13 (7.5 μg/ml) for 60 min and then treated with transferrin-TRITC (50 μg/ml) for 30 min at 37°C. Next, cells were fixed and stained with anti-WAVE (Alexa Fluor 488). Bar, 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Protein Kinase C? and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    doi: 10.1091/mbc.E07-05-0411

    Figure Lengend Snippet: N-WASP or WAVE are not involved in W13 effect. (A) COS1 cells transiently expressing GFP-N-WASP-ΔWA were incubated with W13 (5 μg/ml) for 90 min and EGF (100 ng/ml) for the last 15 min at 37°C, fixed, and immunolabeled with anti-EGFR (Alexa Fluor 594). Bar, 10 μm. (B) NRK cells were preincubated with W13 (7.5 μg/ml) for 60 min and then treated with transferrin-TRITC (50 μg/ml) for 30 min at 37°C. Next, cells were fixed and stained with anti-WAVE (Alexa Fluor 488). Bar, 10 μm.

    Article Snippet: Primary antibodies used were as follows: mouse monoclonal anti-EGFR (American Type Culture Collection, Manassas, VA), mouse monoclonal anti-actin (Valeant Pharmaceuticals, Costa Mesa, CA), rabbit anti-RhoB (Bethyl Laboratories, Montgomery, TX), mouse monoclonal anti-early endosomal antibody (EEA)1 (BD Biosciences Transduction Laboratories, Erembodegem, Belgium), mouse monoclonal anti-p16-Arc (Synaptic Systems, Göttingen, Germany), rabbit anti-WASP family Verprolin-homologous protein (WAVE) and mouse monoclonal anti-cortactin (Upstate Biotechnology, Charlottesville, VA), rabbit anti-green fluorescent protein (GFP) (Abcam, Cambridge, United Kingdom), rabbit anti-PKCδ (Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Expressing, Incubation, Immunolabeling, Staining

    ELOVL4-mediated biosynthesis of VLC-PUFAs is independent of N-glycosylation but requires retention in the ER. A: Western blot showing increased expression of ΔNG compared with WT (left panel) and comparable levels of Δ3His and ΔLys to WT (right panel) in ARPE19 cells. Blots were reprobed for GFP and β-actin. B: ΔNG showed increased levels of 34:5n3 and 36:5n3 elongation products compared with WT in ARPE19 cells supplemented with 20:5n3, correlating with increased expression levels of the protein. ΔLys and Δ3His did not show any detectable level of the elongated VLC-PUFA products. Data are represented as the mean ± SD (n = 3); significance was determined in comparison to WT; * P

    Journal: Journal of Lipid Research

    Article Title: Endoplasmic reticulum microenvironment and conserved histidines govern ELOVL4 fatty acid elongase activity

    doi: 10.1194/jlr.M045443

    Figure Lengend Snippet: ELOVL4-mediated biosynthesis of VLC-PUFAs is independent of N-glycosylation but requires retention in the ER. A: Western blot showing increased expression of ΔNG compared with WT (left panel) and comparable levels of Δ3His and ΔLys to WT (right panel) in ARPE19 cells. Blots were reprobed for GFP and β-actin. B: ΔNG showed increased levels of 34:5n3 and 36:5n3 elongation products compared with WT in ARPE19 cells supplemented with 20:5n3, correlating with increased expression levels of the protein. ΔLys and Δ3His did not show any detectable level of the elongated VLC-PUFA products. Data are represented as the mean ± SD (n = 3); significance was determined in comparison to WT; * P

    Article Snippet: In this study, we used mouse anti-HA (Cell Signaling Technology, Inc., Danvers, MA), rabbit anti-HA (Clonetech, Mountain View, CA), mouse anti-β actin (Abcam, Cambridge, MA), mouse anti-calnexin (Abcam), and rabbit anti-Green Fluorescent Protein (GFP) (Sigma-Aldrich, St. Louis, MO) antibodies.

    Techniques: Western Blot, Expressing