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mruby2 tubulin 6 vector  (Addgene inc)


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    Structured Review

    Addgene inc mruby2 tubulin 6 vector
    CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing <t>mRuby2-Tubulin</t> (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.
    Mruby2 Tubulin 6 Vector, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 86 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mruby2+n1+vector/piRFP670-N1+(Plasmid+%2345457)/pmc06601206-97-10-15
    Average 93 stars, based on 86 article reviews
    mruby2 tubulin 6 vector - by Bioz Stars, 2026-09
    93/100 stars

    Images

    1) Product Images from "Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes * "

    Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

    Journal: Molecular & Cellular Proteomics : MCP

    doi: 10.1074/mcp.RA119.001450

    CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.
    Figure Legend Snippet: CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.

    Techniques Used: Live Cell Imaging, Cell Culture, Immunofluorescence, Immunolabeling

    The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.
    Figure Legend Snippet: The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.

    Techniques Used: Live Cell Imaging, Imaging

    Related Articles

    Plasmid Preparation:

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase.
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. 6 C, cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. Following three days of culture, the cells were pelleted and lysed to prepare genomic DNA for ddPCR, as described above [90 μl of DirectPCR Cell lysis reagent (Viagen) with 1.0 mg/ml proteinase K (Meridian Bioscience) was rotated for 3 h at 55 ◦C then heat-inactivated for 45 min at 85 ◦C].

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. , cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. A FACSAria IIu flow cytometer (BD Biosciences) was used to sort the brightest ∼20% of piRFP670 + live cells (excitation 635 nm, emission 780/60 nm).

    Article Title: A Reporter Platform to Study Therapy‐Induced Senescence in Live Cancer Cells
    Article Snippet: .. Dronpa‐LaminB1‐10 was a gift from Michael Davidson, used for the creation of the SRbr, SRbfr, SRsh, SRf and SRs constructs (Addgene plasmid #57 282); mCherry‐LaminA‐C‐18 was a gift from Michael Davidson (Addgene plasmid #55 068); pHRM‐NLS‐dCas9‐GFP11×7‐NLS‐P2A‐BFP‐dWPRE was a gift from Bo Huang for the creation of the SRs construct (Addgene plasmid #70 224) [ ] ; piRFP670‐N1 was a gift from Vladislav Verkhusha to create the SRbfr construct (Addgene plasmid #45 457) and pmScarlet‐i_C1 was a gift from Dorus Gadella for the SRbr, SRsh, SRf and SRs constructs (Addgene plasmid #85 044). .. [ ] pMK243 was a gift from Masato Kanemaki (Addgene plasmid #72 835) [ ] to create the SRsh construct.

    Article Title: Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice
    Article Snippet: .. HEK293 cells were co-transfected with an expression plasmid encoding mGlu1 and piRFP670-N1 (a gift from Dr. V. Verkhusha, Addgene plasmid #45457) as a transfection marker. ..

    Article Title: Cas9 variants and uses thereof
    Article Snippet: .. Separate wells were transfected with 1 μg of a near-infrared iRFP670 (Addgene plasmid 45457) 32 as a transfection control. ..

    Article Title: A Reporter Platform to Study Therapy-Induced Senescence in Live Cancer Cells.
    Article Snippet: .. Dronpa-LaminB1-10 was a gift from Michael Davidson, used for the creation of the SRbr, SRbfr, SRsh, SRf and SRs constructs (Addgene plasmid #57 282); mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid #55 068); pHRM-NLS-dCas9-GFP11×7NLS-P2A-BFP-dWPRE was a gift from Bo Huang for the creation of the SRs construct (Addgene plasmid #70 224)[59]; piRFP670-N1 was a gift from Vladislav Verkhusha to create the SRbfr construct (Addgene plasmid #45 457) and pmScarlet-i_C1 was a gift from Dorus Gadella for the SRbr, SRsh, SRf and SRs constructs (Addgene plasmid #85 044). .. [60] pMK243 was a gift from Masato Kanemaki (Addgene plasmid #72 835)[61] to create the SRsh construct.

    Article Title: Novel Kinesin Family Member 1A Variants Linked to Atypical Parkinsonism Elicit Altered Neuronal Transactive Response DNA Binding Protein 43 kDa Interactions and Dendritic Atrophy
    Article Snippet: The human synapsin I promoter (hSyn) was PCR amplified from pLV-hSyn-RFP (Addgene, Watertown, MA; catalog number 22909). .. The fragment was then inserted into piRFP670-N1 (Addgene; 45457) at AflIII and HindIII restriction enzyme sites to produce base vector hSyn-iRFP670. .. The hSyn::iRFP fragment was PCR amplified from this base vector and transferred into lentiviral backbone (Addgene; 36084) at BsrGI and NdeI restriction enzyme sites.

    Control:

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase.
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. 6 C, cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. Following three days of culture, the cells were pelleted and lysed to prepare genomic DNA for ddPCR, as described above [90 μl of DirectPCR Cell lysis reagent (Viagen) with 1.0 mg/ml proteinase K (Meridian Bioscience) was rotated for 3 h at 55 ◦C then heat-inactivated for 45 min at 85 ◦C].

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. , cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. A FACSAria IIu flow cytometer (BD Biosciences) was used to sort the brightest ∼20% of piRFP670 + live cells (excitation 635 nm, emission 780/60 nm).

    Article Title: Cas9 variants and uses thereof
    Article Snippet: .. Separate wells were transfected with 1 μg of a near-infrared iRFP670 (Addgene plasmid 45457) 32 as a transfection control. ..

    Cell Culture:

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase.
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. 6 C, cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. Following three days of culture, the cells were pelleted and lysed to prepare genomic DNA for ddPCR, as described above [90 μl of DirectPCR Cell lysis reagent (Viagen) with 1.0 mg/ml proteinase K (Meridian Bioscience) was rotated for 3 h at 55 ◦C then heat-inactivated for 45 min at 85 ◦C].

    Article Title: Gene-sized DNA insertion at genomic safe harbors in human cells using a site-directed transposase
    Article Snippet: .. Using the final optimal ratio that was determined in the experiment shown in Fig. , cells were co-transfected with 280 ng of ALFA MLT m12 helper plasmid or indicated control helper plasmid, 60 ng of each of rDNA1 NbALFA TALE fusion plasmids, 1980 ng of donor plasmid, and 20 ng of piRFP670-N1 plasmid (Addgene), which constitutively expresses iRFP670, and cultured for 4 days. .. A FACSAria IIu flow cytometer (BD Biosciences) was used to sort the brightest ∼20% of piRFP670 + live cells (excitation 635 nm, emission 780/60 nm).

    Construct:

    Article Title: A Reporter Platform to Study Therapy‐Induced Senescence in Live Cancer Cells
    Article Snippet: .. Dronpa‐LaminB1‐10 was a gift from Michael Davidson, used for the creation of the SRbr, SRbfr, SRsh, SRf and SRs constructs (Addgene plasmid #57 282); mCherry‐LaminA‐C‐18 was a gift from Michael Davidson (Addgene plasmid #55 068); pHRM‐NLS‐dCas9‐GFP11×7‐NLS‐P2A‐BFP‐dWPRE was a gift from Bo Huang for the creation of the SRs construct (Addgene plasmid #70 224) [ ] ; piRFP670‐N1 was a gift from Vladislav Verkhusha to create the SRbfr construct (Addgene plasmid #45 457) and pmScarlet‐i_C1 was a gift from Dorus Gadella for the SRbr, SRsh, SRf and SRs constructs (Addgene plasmid #85 044). .. [ ] pMK243 was a gift from Masato Kanemaki (Addgene plasmid #72 835) [ ] to create the SRsh construct.

    Article Title: A Reporter Platform to Study Therapy-Induced Senescence in Live Cancer Cells.
    Article Snippet: .. Dronpa-LaminB1-10 was a gift from Michael Davidson, used for the creation of the SRbr, SRbfr, SRsh, SRf and SRs constructs (Addgene plasmid #57 282); mCherry-LaminA-C-18 was a gift from Michael Davidson (Addgene plasmid #55 068); pHRM-NLS-dCas9-GFP11×7NLS-P2A-BFP-dWPRE was a gift from Bo Huang for the creation of the SRs construct (Addgene plasmid #70 224)[59]; piRFP670-N1 was a gift from Vladislav Verkhusha to create the SRbfr construct (Addgene plasmid #45 457) and pmScarlet-i_C1 was a gift from Dorus Gadella for the SRbr, SRsh, SRf and SRs constructs (Addgene plasmid #85 044). .. [60] pMK243 was a gift from Masato Kanemaki (Addgene plasmid #72 835)[61] to create the SRsh construct.

    Expressing:

    Article Title: Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice
    Article Snippet: .. HEK293 cells were co-transfected with an expression plasmid encoding mGlu1 and piRFP670-N1 (a gift from Dr. V. Verkhusha, Addgene plasmid #45457) as a transfection marker. ..

    Transfection:

    Article Title: Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice
    Article Snippet: .. HEK293 cells were co-transfected with an expression plasmid encoding mGlu1 and piRFP670-N1 (a gift from Dr. V. Verkhusha, Addgene plasmid #45457) as a transfection marker. ..

    Article Title: Cas9 variants and uses thereof
    Article Snippet: .. Separate wells were transfected with 1 μg of a near-infrared iRFP670 (Addgene plasmid 45457) 32 as a transfection control. ..

    Marker:

    Article Title: Native receptor-targeted chemogenetics enables cell-type-specific inhibition of endogenous receptors in freely moving mice
    Article Snippet: .. HEK293 cells were co-transfected with an expression plasmid encoding mGlu1 and piRFP670-N1 (a gift from Dr. V. Verkhusha, Addgene plasmid #45457) as a transfection marker. ..



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    Image Search Results


    CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

    doi: 10.1074/mcp.RA119.001450

    Figure Lengend Snippet: CLASP2 and G2L1 colocalize in 3T3-L1 adipocytes. A–B, Live-cell imaging of adipocytes cultured in complete media coexpressing mRuby2-Tubulin (magenta) and GFP-CLASP2-HA (A, green) or mCherry-G2L1-myc (B, green). Live cells were imaged using TIRFM on a 2-s acquisition interval. Time series images to the right of the whole cell image are used to highlight +TIP dynamics within the indicated ROI. C, Immunofluorescence images of adipocytes cultured in complete media cooverexpressing GFP-CLASP2-HA (green) and mCherry-G2L1-myc (magenta). Cells were fixed and immunolabeled for tubulin (inverted white). Bottom row is magnified ROI. ROI, region of interest. Scale bar = 20 μm.

    Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

    Techniques: Live Cell Imaging, Cell Culture, Immunofluorescence, Immunolabeling

    The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Insulin Induces Microtubule Stabilization and Regulates the Microtubule Plus-end Tracking Protein Network in Adipocytes *

    doi: 10.1074/mcp.RA119.001450

    Figure Lengend Snippet: The effect of insulin on CLASP2 +TIP dynamics. Live-cell imaging of adipocytes serum-starved for one hour and subsequently stimulated with 100 nm insulin. Live cells were imaged using TIRFM on a two-second acquisition interval. A, Single frame of live-cell imaging of adipocytes coexpressing GFP-CLASP2 (green) and mRuby2-Tubulin (magenta) at basal state (top row) or 10 mins (bottom row) following insulin stimulation. CLASP2 is displayed in inverted white to highlight +TIP density. B, Quantification of CLASP2-containing +TIP density per unit area (μm2) in adipocytes at basal state or 10 mins following insulin stimulation. Percent increase in CLASP2-containing +TIP density is indicated to the right. Statistical comparison made by paired parametric t test, n = 5 cells. C, Temporally color-coded projection of GFP-CLASP2 localization during a 30 s live-cell imaging interval. The length and extent of color overlap of time-projected CLASP2 localization indicates reduced displacement and hence lower velocity of CLASP2-containing +TIPs during the imaging interval in adipocytes after 10 mins of insulin stimulation. D, Quantification of CLASP2-containing +TIP velocity in adipocytes at basal state or 10 mins following insulin stimulation indicates reduced velocity of CLASP2-containing +TIPS after insulin treatment. Statistical comparison made by unpaired parametric t test, n = 123–125 CLASP2-containing +TIPS from five cells. Scale bars = 5 μm. E, Image of entire cells in the basal state (left panel) or 8 mins post insulin treatment (right panel) extracted from supplemental Video 2. Time series images under the whole cell image are used to highlight insulin-stimulated +TIP dynamics within the indicated ROI. F, Time series extracted from supplemental Video 2 of the indicated ROIs at either the basal state or at 4 mins post insulin treatment to present an example of changing CLASP2 microtubule plus-end dynamics with insulin stimulation. G, Live cell CLASP2-containing +TIP dynamics of the ROI extracted from supplemental Video 2 were captured in the basal state followed by stimulation with insulin. Each insulin-stimulated CLASP2-containing +TIP trail length time point was compared against the basal CLASP2-containing +TIP trail length to test for significant differences. t test; *p ≤ 0.05, **p ≤ 0.01. Red circles represent outlier data points. ROI, region of interest. BAS, basal. INS, insulin. Scale bar = 10 μm.

    Article Snippet: To generate pLenti-iRFP670-Tubulin, tubulin was first subcloned out of the mRuby2-Tubulin-6 vector and into piRFP670-N1 (Addgene #45457, a gift from Vladislav Verkhusha). iRFP670-Tubulin was then subcloned out of the piRFP670-Tubulin vector and into the pLenti backbone to generate pLenti-iRFP670-Tubulin.

    Techniques: Live Cell Imaging, Imaging