Structured Review

Rockland Immunochemicals goat anti green fluorescent protein
Goat Anti Green Fluorescent Protein, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/goat anti green fluorescent protein/product/Rockland Immunochemicals
Average 97 stars, based on 1 article reviews
Price from $9.99 to $1999.99
goat anti green fluorescent protein - by Bioz Stars, 2022-10
97/100 stars

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    Rockland Immunochemicals goat anti gfp
    CaMKIIδB localizes to the nucleus and inhibits <t>HDAC4</t> in CPCs undergoing cardiogenic commitment. A , immunoblot of total HDAC4 and protein levels in whole, cytosolic and nuclear fractions of CPCs treated with GM, −Dex, or + Dex supplemented medium. B , quantitation of total HDAC4. C , immunoblot of pHDAC4 (s632) protein levels in whole, cytosolic, and nuclear fractions of CPCs treated with GM, −Dex, or + Dex-supplemented medium. D , quantitation of pHDAC4 (s632). Immunoblots are presented as a fold change relative to CPCs in GM after normalization to GAPDH or Lamin A (C terminus). E , lentiviral constructs to establish CPCe and CPCeδB lines ( top ). CPCeδB lines overexpress CaMKIIδB as well as the HA tag. CPCe overexpress eGFP. GAPDH was probed as a loading control ( bottom ). F , CPCe ( top ) and CPCeδB ( bottom ) fluorescent images. Cells were stained with antibodies toward <t>GFP</t> and CAMKIIδ. Cell morphology and nuclei were visualized by staining for tubulin and with DAPI, respectively. G , CPCe and CPCeδB lines probed for total HDAC4 levels by immunoblot and H , quantitation in whole cell lysates, cytosolic and nuclear fractions. I , phosphorylated HDAC4 on the serine 632 protein levels in CPCe or CPCeδB by immunoblot and J , quantitated from whole cell lysates,cytosolic, and nuclear fractions. Immunoblots are probed with GAPDH to normalize for protein loading of the whole and cytosolic lysates. Lamin A (C terminus) antibody was utilized to normalize for loading of the nuclear fraction. Total and phosphorylated HDAC4 are represented as a fold change relative to CPCe. *, p
    Goat Anti Gfp, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti gfp/product/Rockland Immunochemicals
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti gfp - by Bioz Stars, 2022-10
    98/100 stars
      Buy from Supplier

    90
    Rockland Immunochemicals gfp goat antibody biotin conjugated
    EHD2 is not required for normal primary ciliogenesis. (A–H) Representative micrographs of NIH3T3 cells that were engineered by CRISPR/Cas9 to express endogenous levels of EHD2 tagged with <t>GFP</t> (EHD2‐GFP) depicting primary cilia labeled with antibodies against acetylated tubulin (red) and DAPI stain (blue). CRISPR/Cas9 gene‐edited NIH3T3 EHD2‐GFP cells were either mock‐treated with transfection reagent (A, inset in B–D), or transfected with EHD2 siRNA (E, inset in F–H) for 48 h, fixed and immunostained with DAPI and an acetylated tubulin <t>antibody</t> prior to imaging. (I) Validation of EHD2 siRNA efficacy by immunoblot analysis. (J) Graph depicting the percentage of ciliated cells in mock‐treated and EHD2 knock‐down NIH3T3 EHD2‐GFP cells. Error bars denote standard deviation and p values for each experiment were determined by an independent two‐tailed t test. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bars, 10 μm. n.s. = not significant (consensus p > 0.05)
    Gfp Goat Antibody Biotin Conjugated, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gfp goat antibody biotin conjugated/product/Rockland Immunochemicals
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    gfp goat antibody biotin conjugated - by Bioz Stars, 2022-10
    90/100 stars
      Buy from Supplier

    90
    Rockland Immunochemicals polyclonal goat anti gfp antibody
    Study design and MVF characterization. Study design (a): Combined lymphatic ablation by means of irradiation (top left) and popliteal lymphadenectomy 10 days later (top right, red frame = lymphadenectomy site). For identification of the popliteal lymphatic system, hindlimbs were injected with methylene blue. Three days after lymphadenectomy, MVF (red)-enriched collagen hydrogel (green) was injected in the popliteal defect (bottom right). On day 14 and 28, repetitive MR lymphography using the nanoparticle AguIX was performed to evaluate lymphatic regeneration (bottom left). HE-stained (b) and immunohistochemical (c–e) sections of in vitro suspended collagen/MVF hydrogel, revealing LYVE-1 + <t>/GFP</t> + lymphatic (arrowhead) and LYVE-1 − <t>/GFP</t> + blood vessel fragments (asterisk). Scale bars: (b) = 50 µm, (c–e) = 30 µm. Quantitative analysis of mRNA expression levels in normoxic (N) and hypoxic (H) MVF (f–k). VEGF-A (f), IGF-1 (g), Prox1 (h), LYVE-1 (i), VEGF-C (j), and VEGF-D (k) mRNA levels are expressed in % normoxia ( n = 3). Mean ± SEM. * p
    Polyclonal Goat Anti Gfp Antibody, supplied by Rockland Immunochemicals, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/polyclonal goat anti gfp antibody/product/Rockland Immunochemicals
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    polyclonal goat anti gfp antibody - by Bioz Stars, 2022-10
    90/100 stars
      Buy from Supplier

    Image Search Results


    CaMKIIδB localizes to the nucleus and inhibits HDAC4 in CPCs undergoing cardiogenic commitment. A , immunoblot of total HDAC4 and protein levels in whole, cytosolic and nuclear fractions of CPCs treated with GM, −Dex, or + Dex supplemented medium. B , quantitation of total HDAC4. C , immunoblot of pHDAC4 (s632) protein levels in whole, cytosolic, and nuclear fractions of CPCs treated with GM, −Dex, or + Dex-supplemented medium. D , quantitation of pHDAC4 (s632). Immunoblots are presented as a fold change relative to CPCs in GM after normalization to GAPDH or Lamin A (C terminus). E , lentiviral constructs to establish CPCe and CPCeδB lines ( top ). CPCeδB lines overexpress CaMKIIδB as well as the HA tag. CPCe overexpress eGFP. GAPDH was probed as a loading control ( bottom ). F , CPCe ( top ) and CPCeδB ( bottom ) fluorescent images. Cells were stained with antibodies toward GFP and CAMKIIδ. Cell morphology and nuclei were visualized by staining for tubulin and with DAPI, respectively. G , CPCe and CPCeδB lines probed for total HDAC4 levels by immunoblot and H , quantitation in whole cell lysates, cytosolic and nuclear fractions. I , phosphorylated HDAC4 on the serine 632 protein levels in CPCe or CPCeδB by immunoblot and J , quantitated from whole cell lysates,cytosolic, and nuclear fractions. Immunoblots are probed with GAPDH to normalize for protein loading of the whole and cytosolic lysates. Lamin A (C terminus) antibody was utilized to normalize for loading of the nuclear fraction. Total and phosphorylated HDAC4 are represented as a fold change relative to CPCe. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Nuclear Calcium/Calmodulin-dependent Protein Kinase II Signaling Enhances Cardiac Progenitor Cell Survival and Cardiac Lineage Commitment *

    doi: 10.1074/jbc.M115.657775

    Figure Lengend Snippet: CaMKIIδB localizes to the nucleus and inhibits HDAC4 in CPCs undergoing cardiogenic commitment. A , immunoblot of total HDAC4 and protein levels in whole, cytosolic and nuclear fractions of CPCs treated with GM, −Dex, or + Dex supplemented medium. B , quantitation of total HDAC4. C , immunoblot of pHDAC4 (s632) protein levels in whole, cytosolic, and nuclear fractions of CPCs treated with GM, −Dex, or + Dex-supplemented medium. D , quantitation of pHDAC4 (s632). Immunoblots are presented as a fold change relative to CPCs in GM after normalization to GAPDH or Lamin A (C terminus). E , lentiviral constructs to establish CPCe and CPCeδB lines ( top ). CPCeδB lines overexpress CaMKIIδB as well as the HA tag. CPCe overexpress eGFP. GAPDH was probed as a loading control ( bottom ). F , CPCe ( top ) and CPCeδB ( bottom ) fluorescent images. Cells were stained with antibodies toward GFP and CAMKIIδ. Cell morphology and nuclei were visualized by staining for tubulin and with DAPI, respectively. G , CPCe and CPCeδB lines probed for total HDAC4 levels by immunoblot and H , quantitation in whole cell lysates, cytosolic and nuclear fractions. I , phosphorylated HDAC4 on the serine 632 protein levels in CPCe or CPCeδB by immunoblot and J , quantitated from whole cell lysates,cytosolic, and nuclear fractions. Immunoblots are probed with GAPDH to normalize for protein loading of the whole and cytosolic lysates. Lamin A (C terminus) antibody was utilized to normalize for loading of the nuclear fraction. Total and phosphorylated HDAC4 are represented as a fold change relative to CPCe. *, p

    Article Snippet: Next day blots were washed with TBST buffer and incubated in secondary antibodies in milk for 1.5 h. Primary antibodies for Western blot are as follows: rabbit anti-CaMKIIδ (1:15000; UC Davis, Dr. Donald Bers), mouse anti-HDAC4 (1:1,000; Cell Signaling #5392)rabbit anti-phospho-HDAC4 (Ser-632)/HDAC5 (Ser-661)/HDAC7 (Ser-486) (1:1000; Cell Signaling #3424) goat anti-GFP (1:1000; Rockland 600-101-215), mouse anti-HA tag (1:200; Santa Cruz Biotechnology, Inc. sc-7392), mouse anti-p16 (1:200, Santa Cruz Biotechnology, Inc. F-9 and F-12 clones sc-55600, sc-1661), mouse anti-p53 (1:500, Abcam ab26), rabbit anti-α-sarcomeric actinin (1:500; Abcam ab72592), mouse anti-α-smooth muscle actin (1:1000; Sigma Aldrich a2547), rabbit anti-Tie2 (1:100; Santa Cruz Biotechnology Inc. sc-9026), mouse anti-mouse β-actin (1:500; Santa Cruz Biotechnology Inc sc-81178), rabbit anti-Mef2c (1:500; Cell Signaling #5030), rabbit anti-Cyclin B1 (1:500; Cell Signaling #12231), rabbit anti-Cyclin D1 (1:1000; Cell Signaling #2978), rabbit anti-phospho histone H3 (1:1000, Life Technologies 44–1190G), mouse anti-GAPDH (1:3000; Millipore AB2302), and rabbit anti-Lamin A (C-terminal) (1:1000; Sigma Aldrich L1293).

    Techniques: Quantitation Assay, Western Blot, Construct, Staining

    p62 affects the timing of ubiquitination. (A,B) Time‐lapse images of pH rodo and GFP ‐Ubwt fluorescence around a single pH rodo bead in MEF cells. Images were obtained every minute for ~ 30 min. The panels show representative images of pH rodo and GFP ‐Ubwt fluorescence in p62‐ KO / GFP ‐Ubwt MEF cells (A) and p62‐ KO / GFP ‐Ubwt/p62 MEF cells ectopically expressing p62 (B). Scale bar, 2 μm. A roundish bleb‐like structure next to the beads is often observed in the MEF cell lines used in this study when the beads are in the acidic endosome. (C) Statistical analysis was performed for the timing of GFP ‐signal accumulation around the beads after the loss of pH rodo signals in the GFP ‐Ubwt MEF cells that endogenously express p62 (lane 1; this was copied from Fig. 1 C for ease of comparison), p62‐ KO / GFP ‐Ubwt cells not expressing p62 (lane 2), p62‐ KO / GFP ‐Ubwt/p62 cells that ectopically expressing p62 (lane 3), p62‐ KO / GFP ‐Ubwt/p62S405A cells ectopically expressing the p62S405A mutant (lane 4), and p62‐ KO / GFP ‐Ubwt/p62S405E cells ectopically expressing the p62S405E mutant (lane 5). The median time values were 3 min for GFP ‐Ubwt ( n = 26 beads), 10 min for p62‐ KO / GFP ‐Ubwt ( n = 33 beads), 4 min for p62‐ KO / GFP ‐Ubwt/p62 ( n = 30 beads), 10 min for p62‐ KO / GFP ‐Ubwt/p62S405A ( n = 36 beads), and 4 min for p62‐ KO / GFP ‐Ubwt/p62S405E ( n = 39 beads). Statistical differences ( P

    Journal: FEBS Open Bio

    Article Title: p62/ SQSTM1 promotes rapid ubiquitin conjugation to target proteins after endosome rupture during xenophagy

    doi: 10.1002/2211-5463.12385

    Figure Lengend Snippet: p62 affects the timing of ubiquitination. (A,B) Time‐lapse images of pH rodo and GFP ‐Ubwt fluorescence around a single pH rodo bead in MEF cells. Images were obtained every minute for ~ 30 min. The panels show representative images of pH rodo and GFP ‐Ubwt fluorescence in p62‐ KO / GFP ‐Ubwt MEF cells (A) and p62‐ KO / GFP ‐Ubwt/p62 MEF cells ectopically expressing p62 (B). Scale bar, 2 μm. A roundish bleb‐like structure next to the beads is often observed in the MEF cell lines used in this study when the beads are in the acidic endosome. (C) Statistical analysis was performed for the timing of GFP ‐signal accumulation around the beads after the loss of pH rodo signals in the GFP ‐Ubwt MEF cells that endogenously express p62 (lane 1; this was copied from Fig. 1 C for ease of comparison), p62‐ KO / GFP ‐Ubwt cells not expressing p62 (lane 2), p62‐ KO / GFP ‐Ubwt/p62 cells that ectopically expressing p62 (lane 3), p62‐ KO / GFP ‐Ubwt/p62S405A cells ectopically expressing the p62S405A mutant (lane 4), and p62‐ KO / GFP ‐Ubwt/p62S405E cells ectopically expressing the p62S405E mutant (lane 5). The median time values were 3 min for GFP ‐Ubwt ( n = 26 beads), 10 min for p62‐ KO / GFP ‐Ubwt ( n = 33 beads), 4 min for p62‐ KO / GFP ‐Ubwt/p62 ( n = 30 beads), 10 min for p62‐ KO / GFP ‐Ubwt/p62S405A ( n = 36 beads), and 4 min for p62‐ KO / GFP ‐Ubwt/p62S405E ( n = 39 beads). Statistical differences ( P

    Article Snippet: Proteins were transferred to polyvinylidene fluoride membranes and probed using rabbit anti‐GFP (600‐401‐215; Rockland Immunochemicals, Pottstown, PA, USA), anti‐p62/SQSTMI (P0067; Sigma‐Aldrich), or anti‐glyceraldehyde 3‐phosphate dehydrogenase (anti‐GAPDH) antibody (14C10; Cell Signaling Technology, Danvers, MA, USA) and secondary antibodies conjugated to horseradish peroxidase (NA9340; GE Healthcare Life Sciences).

    Techniques: Fluorescence, Expressing, Mutagenesis

    EHD2 is not required for normal primary ciliogenesis. (A–H) Representative micrographs of NIH3T3 cells that were engineered by CRISPR/Cas9 to express endogenous levels of EHD2 tagged with GFP (EHD2‐GFP) depicting primary cilia labeled with antibodies against acetylated tubulin (red) and DAPI stain (blue). CRISPR/Cas9 gene‐edited NIH3T3 EHD2‐GFP cells were either mock‐treated with transfection reagent (A, inset in B–D), or transfected with EHD2 siRNA (E, inset in F–H) for 48 h, fixed and immunostained with DAPI and an acetylated tubulin antibody prior to imaging. (I) Validation of EHD2 siRNA efficacy by immunoblot analysis. (J) Graph depicting the percentage of ciliated cells in mock‐treated and EHD2 knock‐down NIH3T3 EHD2‐GFP cells. Error bars denote standard deviation and p values for each experiment were determined by an independent two‐tailed t test. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bars, 10 μm. n.s. = not significant (consensus p > 0.05)

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis. Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis

    doi: 10.1111/tra.12845

    Figure Lengend Snippet: EHD2 is not required for normal primary ciliogenesis. (A–H) Representative micrographs of NIH3T3 cells that were engineered by CRISPR/Cas9 to express endogenous levels of EHD2 tagged with GFP (EHD2‐GFP) depicting primary cilia labeled with antibodies against acetylated tubulin (red) and DAPI stain (blue). CRISPR/Cas9 gene‐edited NIH3T3 EHD2‐GFP cells were either mock‐treated with transfection reagent (A, inset in B–D), or transfected with EHD2 siRNA (E, inset in F–H) for 48 h, fixed and immunostained with DAPI and an acetylated tubulin antibody prior to imaging. (I) Validation of EHD2 siRNA efficacy by immunoblot analysis. (J) Graph depicting the percentage of ciliated cells in mock‐treated and EHD2 knock‐down NIH3T3 EHD2‐GFP cells. Error bars denote standard deviation and p values for each experiment were determined by an independent two‐tailed t test. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bars, 10 μm. n.s. = not significant (consensus p > 0.05)

    Article Snippet: 2.2 AntibodiesThe following antibodies were used (also see Table ): Rabbit anti‐EHD1 (Abcam, ab109311), rabbit anti‐EHD4, Rabbit anti‐acetyl‐α‐tubulin (Lys40) (D20G3) (Cell Signaling, 5335), mouse anti‐acetylated tubulin (Sigma‐Aldrich, T7451), rabbit anti‐CP110 (ProteinTech, 12780‐1‐AP), rabbit anti‐ARL13B (ProteinTech, 17711‐1‐AP), mouse anti‐GFP (Roche, 11814460001), mouse anti‐pan actin (Novus, NB600‐535), mouse anti‐GAPDH‐HRP (ProteinTech, HRP‐60004), donkey anti‐mouse‐HRP (Jackson, 715‐035‐151), mouse anti‐rabbit IgG light chain‐HRP (Jackson, 211‐032‐171), DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride) (Molecular Probes, D1306), biotin‐conjugated goat anti‐GFP (Rockland, 600‐106‐215), Alexa‐fluor 488‐conjugated streptavidin (Molecular Probes, S11223), Alexa‐fluor 488‐conjugated goat anti‐mouse (Molecular Probes, A11029), Alexa‐fluor 568‐conjugated goat anti‐rabbit (Molecular Probes, A11036), Alexa‐fluor 568‐conjugated goat anti‐mouse (Molecular Probes, A11031), Alexa‐fluor 633‐conjugated goat anti‐rabbit (Molecular Probes, A21070).

    Techniques: CRISPR, Labeling, Staining, Transfection, Imaging, Standard Deviation, Two Tailed Test, Derivative Assay

    EHD1 P446S and E470W do not rescue ciliogenesis. (A–L) Representative micrographs depicting primary cilia labeled with acetylated tubulin (red), GFP‐EHD1 (green) and DAPI stain (blue) in NIH3T3 EHD1‐KO cells that were mock‐treated (no transfection), or transfected with GFP‐EHD1 WT, GFP‐EHD1 P446S, or GFP‐EHD1 E470W. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), transfected with GFP‐EHD1 P446S (G–I), or transfected with GFP‐EHD1 E470W (J–L) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (M) Validation of GFP‐EHD1 transfection efficiency by immunoblot analysis. (N) Graph depicting the percentage of ciliated cells in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. (O) Graph illustrating the percent of cells with EHD1 localized to the primary cilium or centrosome in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. Error bars denote standard deviation and p values for each experiment were determined by one‐way ANOVA. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bar, 10 μm. (i) consensus p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis. Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis

    doi: 10.1111/tra.12845

    Figure Lengend Snippet: EHD1 P446S and E470W do not rescue ciliogenesis. (A–L) Representative micrographs depicting primary cilia labeled with acetylated tubulin (red), GFP‐EHD1 (green) and DAPI stain (blue) in NIH3T3 EHD1‐KO cells that were mock‐treated (no transfection), or transfected with GFP‐EHD1 WT, GFP‐EHD1 P446S, or GFP‐EHD1 E470W. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), transfected with GFP‐EHD1 P446S (G–I), or transfected with GFP‐EHD1 E470W (J–L) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (M) Validation of GFP‐EHD1 transfection efficiency by immunoblot analysis. (N) Graph depicting the percentage of ciliated cells in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. (O) Graph illustrating the percent of cells with EHD1 localized to the primary cilium or centrosome in mock‐treated, GFP‐EHD1 WT, GFP‐EHD1 P446S and GFP‐EHD1 E470W cells. Error bars denote standard deviation and p values for each experiment were determined by one‐way ANOVA. All three experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the three experiments. Micrographs are representative orthogonal projections from three independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bar, 10 μm. (i) consensus p

    Article Snippet: 2.2 AntibodiesThe following antibodies were used (also see Table ): Rabbit anti‐EHD1 (Abcam, ab109311), rabbit anti‐EHD4, Rabbit anti‐acetyl‐α‐tubulin (Lys40) (D20G3) (Cell Signaling, 5335), mouse anti‐acetylated tubulin (Sigma‐Aldrich, T7451), rabbit anti‐CP110 (ProteinTech, 12780‐1‐AP), rabbit anti‐ARL13B (ProteinTech, 17711‐1‐AP), mouse anti‐GFP (Roche, 11814460001), mouse anti‐pan actin (Novus, NB600‐535), mouse anti‐GAPDH‐HRP (ProteinTech, HRP‐60004), donkey anti‐mouse‐HRP (Jackson, 715‐035‐151), mouse anti‐rabbit IgG light chain‐HRP (Jackson, 211‐032‐171), DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride) (Molecular Probes, D1306), biotin‐conjugated goat anti‐GFP (Rockland, 600‐106‐215), Alexa‐fluor 488‐conjugated streptavidin (Molecular Probes, S11223), Alexa‐fluor 488‐conjugated goat anti‐mouse (Molecular Probes, A11029), Alexa‐fluor 568‐conjugated goat anti‐rabbit (Molecular Probes, A11036), Alexa‐fluor 568‐conjugated goat anti‐mouse (Molecular Probes, A11031), Alexa‐fluor 633‐conjugated goat anti‐rabbit (Molecular Probes, A21070).

    Techniques: Labeling, Staining, Transfection, CRISPR, Imaging, Standard Deviation, Derivative Assay

    Ciliogenesis in EHD1 knock‐out cells is rescued by WT EHD1 but not the EHD1 G65R mutant. (A–I) Representative micrographs depicting primary cilia labeled by acetylated tubulin (red) and GFP‐EHD1 (green) and DAPI stain (blue) in EHD1 knock‐out (KO) cells that were either untransfected, or transfected with GFP‐EHD1 WT, or GFP‐EHD1 G65R. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), or transfected with the GFP‐EHD1 G65R mutant (G–I) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (J) Validation of GFP‐EHD1 WT and G65R transfection efficacy by immunoblot analysis. (K) Graph illustrating the corrected total cell fluorescence values for each cell transfected with either GFP‐EHD1 WT or GFP‐EHD1 G65R. (L) Graph depicting the percentage of ciliated cells in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. (M) Graph illustrating the percent of cells where EHD1 is localized to the primary cilium or centrosome in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. Error bars denote standard deviation, and p values for each experiment were determined by one‐way ANOVA. All six experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the six experiments. Micrographs are representative orthogonal projections from six independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bar, 10 μm. n.s. = not significant (consensus p > 0.05). i: p

    Journal: Traffic (Copenhagen, Denmark)

    Article Title: Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis. Differential requirements for the Eps15 homology domain proteins EHD4 and EHD2 in the regulation of mammalian ciliogenesis

    doi: 10.1111/tra.12845

    Figure Lengend Snippet: Ciliogenesis in EHD1 knock‐out cells is rescued by WT EHD1 but not the EHD1 G65R mutant. (A–I) Representative micrographs depicting primary cilia labeled by acetylated tubulin (red) and GFP‐EHD1 (green) and DAPI stain (blue) in EHD1 knock‐out (KO) cells that were either untransfected, or transfected with GFP‐EHD1 WT, or GFP‐EHD1 G65R. CRISPR/Cas9 gene‐edited NIH3T3 EHD1‐KO cells were either mock‐treated with transfection reagent (no transfection) (A–C), transfected with GFP‐EHD1 WT (D–F), or transfected with the GFP‐EHD1 G65R mutant (G–I) for 48 h, fixed and immunostained with DAPI, an anti‐GFP antibody and an acetylated tubulin antibody prior to imaging. (J) Validation of GFP‐EHD1 WT and G65R transfection efficacy by immunoblot analysis. (K) Graph illustrating the corrected total cell fluorescence values for each cell transfected with either GFP‐EHD1 WT or GFP‐EHD1 G65R. (L) Graph depicting the percentage of ciliated cells in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. (M) Graph illustrating the percent of cells where EHD1 is localized to the primary cilium or centrosome in non‐transfected, GFP‐EHD1 WT transfected and GFP‐EHD1 G65R transfected cells. Error bars denote standard deviation, and p values for each experiment were determined by one‐way ANOVA. All six experiments rely on data from 10 images and each experiment is marked by a distinct shape on the graph. A consensus p value was then derived as described previously to assess significant differences between samples from the six experiments. Micrographs are representative orthogonal projections from six independent experiments, with 10 sets of z‐stacks collected for each treatment per experiment. Bar, 10 μm. n.s. = not significant (consensus p > 0.05). i: p

    Article Snippet: 2.2 AntibodiesThe following antibodies were used (also see Table ): Rabbit anti‐EHD1 (Abcam, ab109311), rabbit anti‐EHD4, Rabbit anti‐acetyl‐α‐tubulin (Lys40) (D20G3) (Cell Signaling, 5335), mouse anti‐acetylated tubulin (Sigma‐Aldrich, T7451), rabbit anti‐CP110 (ProteinTech, 12780‐1‐AP), rabbit anti‐ARL13B (ProteinTech, 17711‐1‐AP), mouse anti‐GFP (Roche, 11814460001), mouse anti‐pan actin (Novus, NB600‐535), mouse anti‐GAPDH‐HRP (ProteinTech, HRP‐60004), donkey anti‐mouse‐HRP (Jackson, 715‐035‐151), mouse anti‐rabbit IgG light chain‐HRP (Jackson, 211‐032‐171), DAPI (4′,6‐diamidino‐2‐phenylindole, dihydrochloride) (Molecular Probes, D1306), biotin‐conjugated goat anti‐GFP (Rockland, 600‐106‐215), Alexa‐fluor 488‐conjugated streptavidin (Molecular Probes, S11223), Alexa‐fluor 488‐conjugated goat anti‐mouse (Molecular Probes, A11029), Alexa‐fluor 568‐conjugated goat anti‐rabbit (Molecular Probes, A11036), Alexa‐fluor 568‐conjugated goat anti‐mouse (Molecular Probes, A11031), Alexa‐fluor 633‐conjugated goat anti‐rabbit (Molecular Probes, A21070).

    Techniques: Knock-Out, Mutagenesis, Labeling, Staining, Transfection, CRISPR, Imaging, Fluorescence, Standard Deviation, Derivative Assay

    Study design and MVF characterization. Study design (a): Combined lymphatic ablation by means of irradiation (top left) and popliteal lymphadenectomy 10 days later (top right, red frame = lymphadenectomy site). For identification of the popliteal lymphatic system, hindlimbs were injected with methylene blue. Three days after lymphadenectomy, MVF (red)-enriched collagen hydrogel (green) was injected in the popliteal defect (bottom right). On day 14 and 28, repetitive MR lymphography using the nanoparticle AguIX was performed to evaluate lymphatic regeneration (bottom left). HE-stained (b) and immunohistochemical (c–e) sections of in vitro suspended collagen/MVF hydrogel, revealing LYVE-1 + /GFP + lymphatic (arrowhead) and LYVE-1 − /GFP + blood vessel fragments (asterisk). Scale bars: (b) = 50 µm, (c–e) = 30 µm. Quantitative analysis of mRNA expression levels in normoxic (N) and hypoxic (H) MVF (f–k). VEGF-A (f), IGF-1 (g), Prox1 (h), LYVE-1 (i), VEGF-C (j), and VEGF-D (k) mRNA levels are expressed in % normoxia ( n = 3). Mean ± SEM. * p

    Journal: Journal of Tissue Engineering

    Article Title: Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model

    doi: 10.1177/20417314221109957

    Figure Lengend Snippet: Study design and MVF characterization. Study design (a): Combined lymphatic ablation by means of irradiation (top left) and popliteal lymphadenectomy 10 days later (top right, red frame = lymphadenectomy site). For identification of the popliteal lymphatic system, hindlimbs were injected with methylene blue. Three days after lymphadenectomy, MVF (red)-enriched collagen hydrogel (green) was injected in the popliteal defect (bottom right). On day 14 and 28, repetitive MR lymphography using the nanoparticle AguIX was performed to evaluate lymphatic regeneration (bottom left). HE-stained (b) and immunohistochemical (c–e) sections of in vitro suspended collagen/MVF hydrogel, revealing LYVE-1 + /GFP + lymphatic (arrowhead) and LYVE-1 − /GFP + blood vessel fragments (asterisk). Scale bars: (b) = 50 µm, (c–e) = 30 µm. Quantitative analysis of mRNA expression levels in normoxic (N) and hypoxic (H) MVF (f–k). VEGF-A (f), IGF-1 (g), Prox1 (h), LYVE-1 (i), VEGF-C (j), and VEGF-D (k) mRNA levels are expressed in % normoxia ( n = 3). Mean ± SEM. * p

    Article Snippet: To differentiate between MVF-derived GFP+ and wild-type GFP− blood and lymphatic vessels, sections were stained with the above-mentioned primary and secondary antibodies against CD31 and LYVE-1 and with a polyclonal goat anti-GFP antibody (1:100; Rockland, Limerick, PA).

    Techniques: Irradiation, Injection, Staining, Immunohistochemistry, In Vitro, Expressing

    Popliteal lymphatic vessel density after MVF transplantation. Immunohistochemical detection (a–c) of MVF-derived LYVE-1 + /GFP + lymphatic vessels (arrowheads). Scale bars = 30 µm. Quantification of overall LV density in mm −2 (d). Quantification of zonal LV density in mm −2 (e). Mean ± SEM, n = 8–9, * p

    Journal: Journal of Tissue Engineering

    Article Title: Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model

    doi: 10.1177/20417314221109957

    Figure Lengend Snippet: Popliteal lymphatic vessel density after MVF transplantation. Immunohistochemical detection (a–c) of MVF-derived LYVE-1 + /GFP + lymphatic vessels (arrowheads). Scale bars = 30 µm. Quantification of overall LV density in mm −2 (d). Quantification of zonal LV density in mm −2 (e). Mean ± SEM, n = 8–9, * p

    Article Snippet: To differentiate between MVF-derived GFP+ and wild-type GFP− blood and lymphatic vessels, sections were stained with the above-mentioned primary and secondary antibodies against CD31 and LYVE-1 and with a polyclonal goat anti-GFP antibody (1:100; Rockland, Limerick, PA).

    Techniques: Transplantation Assay, Immunohistochemistry, Derivative Assay

    Popliteal microvessel density after MVF transplantation. HE-stained (a–c) and GFP-stained (d) sections of the popliteal fossa of the control (a), collagen (b) and collagen/MVF (c and d) groups 28 days after lymphadenectomy. Arrowheads in (a–c) = lymphadenectomy scar. Area between dashed lines = epifascial collagen (b) and collagen/MVF (c and d) deposits. Popliteal GFP + MVF-derived blood and lymphatic vessels ((d), arrowheads). Immunohistochemical detection (e–g) of a MVF-derived CD31 + /GFP + microvessel (arrowheads) filled with erythrocytes (asterisks). Quantification of overall MV density in mm −2 (h). Quantification of zonal MV density in mm −2 (i). Mean ± SEM, n = 8–9, * p

    Journal: Journal of Tissue Engineering

    Article Title: Adipose tissue-derived microvascular fragments promote lymphangiogenesis in a murine lymphedema model

    doi: 10.1177/20417314221109957

    Figure Lengend Snippet: Popliteal microvessel density after MVF transplantation. HE-stained (a–c) and GFP-stained (d) sections of the popliteal fossa of the control (a), collagen (b) and collagen/MVF (c and d) groups 28 days after lymphadenectomy. Arrowheads in (a–c) = lymphadenectomy scar. Area between dashed lines = epifascial collagen (b) and collagen/MVF (c and d) deposits. Popliteal GFP + MVF-derived blood and lymphatic vessels ((d), arrowheads). Immunohistochemical detection (e–g) of a MVF-derived CD31 + /GFP + microvessel (arrowheads) filled with erythrocytes (asterisks). Quantification of overall MV density in mm −2 (h). Quantification of zonal MV density in mm −2 (i). Mean ± SEM, n = 8–9, * p

    Article Snippet: To differentiate between MVF-derived GFP+ and wild-type GFP− blood and lymphatic vessels, sections were stained with the above-mentioned primary and secondary antibodies against CD31 and LYVE-1 and with a polyclonal goat anti-GFP antibody (1:100; Rockland, Limerick, PA).

    Techniques: Transplantation Assay, Staining, Derivative Assay, Immunohistochemistry