rabbit anti-gfp Search Results


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  • 99
    Thermo Fisher gfp tag polyclonal antibody
    Cleavage of wild-type (WT) and mutant HIV-1 <t>Tat-GFP</t> proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 <t>polyclonal</t> antibody as indicated.
    Gfp Tag Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8757 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit anti gfp
    The <t>GFAP</t> promoter is astrocyte-specific. (A and B) Two weeks after systemic delivery of either <t>rAAV1/2-GFAP-GFP</t> or rAAV1/2-HBA-GFP, transgene-positive (GFP, green) cells are visible in the FUS-targeted region. Aβ plaque is shown in blue, and GFAP expression in red. (C) At higher magnification in the cortex, the morphology of GFP-positive cells in the rAAV1/2-GFAP-GFP group is consistent and colocalizes with GFAP-positive cells (colocalization, yellow). (D) In the cortex after delivery of rAAV1/2-HBA-GFP, a variety of cell morphologies are visible and are not always colocalized with GFAP. (E and F) In the hippocampus, the same respective trends are seen of GFP-positive cell morphology after delivery of rAAV1/2-GFAP-GFP or rAAV1/2-HBA-GFP. (G) As seen in an orthogonal projection, consistent colocalization between GFP-positive cells and GFAP is verified in the rAAV1/2-GFAP-GFP group. (H) A consistent colocalization between GFP and GFAP is not seen in the orthogonal projection of rAAV1/2-HBA-GFP cells. (I) Quantification of GFP-positive (GFP+) cells are categorized as GFAP+ (astrocyte), or GFAP- (undefined). The percentage of GFP-positive cells that are also GFAP-positive after rAAV1/2-GFAP-GFP delivery indicate almost exclusive transgene expression in astrocytes. (J) The areas containing GFP-positive cells in the cortex and hippocampus after FUS application were not significantly different in size (µm 2 ) between the rAAV1/2-GFAP-GFP and rAAV1/2-HBA-GFP groups (p=0.91). (K) The amount of BBB opening by FUS application can be estimated by the MRI enhancement from background of each focal spot, which was not significantly different between the two rAAV groups (p=0.87). (L) The number of Aβ plaques in the cortical and hippocampal regions containing GFP-positive cells was also not significantly different between rAAV groups (p=0.69). Data is represented as mean ±SEM and (I-L) n=4 animals per group. (K) For MRI enhancement n=12 focal spots from 4 animals, per group. Scale bars: (A and B) 1 mm; (c-f) 100 µm; (G and H) 20 µm.
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    94
    Thermo Fisher rabbit anti gfp
    Distribution of Src1 at the nuclear envelope. ( A ) Localization of <t>GFP-Src1</t> during interphase and mitosis. Mitotic stages are indicated. GFP fluorescence is shown in green, DAPI staining of nuclei in blue, and microtubules in red (labeled with anti-α-tubulin YL1/2 and anti-rat-AlexaFluor 561). Bar, 4 μm. ( B ) Localization of endogenous Src1 labeled with anti-Src1/anti-rabbit AlexaFluor 488 (green), <t>anti-NE81/anti-AlexaFluor</t> 568 (red), and DAPI (blue). Note the uneven distribution of Src1 with accumulations at sites of nucleolar attachment to the nuclear envelope. Nucleoli (arrowheads) are poorly stained with DAPI and visible in phase contrast images as dark structures within the nucleus. ( A, B ) widefield deconvolution microscopy. Specimens were fixed with glutaraldehyde. Maximum intensity projections of thin image stacks through the center of the nucleus are shown. Bar, 5 μm. ( B' ) Quantitative evaluation of anti-Src1 staining intensities in nucleolar (No) vs. non-nucleolar (non-No) regions at the NE. Mean normalized staining intensities ± S.D. are shown ( n = 60 for each region). ( C ) Western blot of cytosolic extracts of wild-type, GFP-Src1 full length, and GFP-Src1 1–646 cells stained with anti-Src1 antibodies and anti-GFP antibodies as indicated. Numbers refer to relative staining intensities, whereby Src1 staining in wild-type cells was set to 1. As protein levels of GFP-Src1 are the same whether stained with anti-Src1 or anti-GFP, the protein level of GFP- Src1 1–646 compared to endogenous Src1 could be deduced. Actin is shown as a loading control.
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    Abcam rabbit anti gfp
    RABGEF1 is recruited to the damaged mitochondria in a ubiquitin-binding dependent manner. ( A ) HeLa cells transiently expressing mChery-Parkin and <t>GFP-mRABGEF1</t> were treated with DMSO or valinomycin for 3 hr followed by <t>immunostaining.</t> The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding.
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    Santa Cruz Biotechnology rabbit anti gfp
    Regulation of autophagy by MC159 and its SH3BP4 binding-deficient mutant. (A and B) Control MCF-7/LC3-EGFP cells or their lentivirally transduced derivatives stably expressing wild-type MC159 or MC159 AXXA(N) were examined under normal culture conditions (A) or after 6 h of starvation (B) in medium lacking amino acids and serum using fluorescence microscopy imaging of nuclear (Hoechst)-, LC3-, and MC159-specific signals, as indicated. (C) Uniform <t>LC3-GFP</t> expression in all cells, equal MC159 expression in the transduced cells, and the lack of SH3BP4 expression in the knockout cells were verified by Western blotting using antibodies against GFP, mCherry, and SH3BP4, respectively. (D) Decrease of <t>p62</t> expression in MCF-7/LC3-EGFP cells (control, C) and their derivatives expressing wild-type MC159 (WT) or MC159 AXXA(N) (AXXA) was examined by Western blotting. The anti-p62 signals were quantified and normalized to levels of the corresponding loading control values (anti-α-tubulin signal) to calculate the relative decrease in p62 expression, which is shown as a bar graph above the blot. (E) Box blot presentation of the data from automated quantification of the number of autophagosomes in the parental MCF-7/LC3-EGFP cells and their SH3BP4 knockout derivatives used as controls (C) or transduced with wild-type MC159 (WT) or MC159 AXXA(N) (AXXA), as indicated. The boxes mark the two middle quartiles of the data points separated by a line showing the median autophagosome count, and the whiskers show the distribution of all values in the upper and lower data point quartiles. The statistical significance of the indicated comparisons is shown as follows: *, P = 6,291 × 10 −93 ; **, P = 5,868 × 10 −101 .
    Rabbit Anti Gfp, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam rabbit anti gfp antibody
    <t>H2A.Z</t> acidic patch is incorporated at lower levels at target genes. ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z WT , H2A.Z AP3 , H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z WT , H2A.Z AP3 , and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z WT , H2A.Z AP3 , and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z WT and H2A.Z AP3 were generated using <t>GFP</t> antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z WT and H2A.Z AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z WT in chromatin fractions compared to H2A.Z AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z WT and H2A.Z AP3 . Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z AP3 . (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z WT and H2A.Z AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z WT and H2A.Z AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.
    Rabbit Anti Gfp Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1183 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit polyclonal anti gfp
    Molecular weight patterns of over-expressed MOS1 fusion in HeLa cells. Proteins extracted from HeLa cells over-expressing V5-MOS1 or <t>MOS1V2-GFP</t> were analyzed by immunoblotting after separation by polyacrylamide gel electrophoresis. V5-MOS1 and MOS1V2-GFP were repectively revealed by first hybridizing a mouse anti-V5 monoclonal antibody or a rabbit <t>polyclonal</t> anti-GFP a mouse a rabbit polyclonal anti-GFP. The filters were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG, followed by development using enhanced chemiluminescence.
    Rabbit Polyclonal Anti Gfp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore anti gfp n terminal antibody
    Lead triggers phase separation of <t>TDP-43</t> and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p
    Anti Gfp N Terminal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 864 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Torrey Pines Biolabs rabbit anti gfp
    Physical interactions between dTgs1 and the SMN complex. Proteins co-purifying with dTgs1 or Smn were identified by affinity purification (AP) using <t>GFP-TRAP</t> beads, followed by mass spectrometry (MS) and stringent filtering (see Materials and Methods for details). AP/MS was carried out using 0–3 hr embryos from mothers expressing UAS-GFP-dTgs1 driven by Actin-Gal4 or Smn-GFP under the control of the tubulin promoter. (A) Efficiency of GFP-TRAP-mediated AP assayed using an anti-GFP antibody (T, total protein extract; I, input (10%); S supernatant; IP, immunoprecipitate). (B) Schematic representations of the snRNP maturation pathway; note that in Drosophila there are only four Gemin proteins. See Introduction for a detailed description of this pathway. (C, D) dTgs1 (C) and Smn (D) interacting proteins. All protein IDs conforming to stringent filtering (see Materials and Methods ) are shown in the Tables. Mean area corresponds to top 3 protein quantification (T3PQ), the mean of the three highest abundance peptides identified for each protein. The complete lists of the <t>Tgs1-</t> and Smn-interacting proteins are shown in S1 and S2 Tables.
    Rabbit Anti Gfp, supplied by Torrey Pines Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam rabbit polyclonal anti gfp
    Diffusion of <t>GFP</t> takes a random route via the NPC. Representative TEM micrographs of high-pressure frozen and freeze-substituted yeast expressing unconjugated GFP immunolabelled with <t>polyclonal</t> anti-GFP primary antibody and anti-rabbit secondary antibody
    Rabbit Polyclonal Anti Gfp, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti gfp
    BDFN overexpression rescues spine phenotype due to eEF2K knock-down. A , Neurons were transfected with <t>GFP,</t> <t>BDNF,</t> sieEF2K, or sieEF2K+BDNF vectors on DIV10, fixed on DIV18, and stained with BDNF antibody. BDNF coexpression increased the BDNF signal along the dendrites and positively affected spine number and morphology in comparison with sieEF2K alone. B–D , Mean length, width and number of dendritic spines (±SEM) in neurons transfected with the indicated constructs; > 10 transfected neurons (corresponding to > 5000 μm in dendrite length), were measured for every condition; * p
    Rabbit Anti Gfp, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gfp tag antibody
    Transcription factor Brn3b promoted an increase in the levels of <t>p-AKT</t> in retinas of rats injected with rAAV-hSyn-Brn3b. A : Immunostaining for p-AKT (pseudogreen), βIII-tubulin (pseudored) expression in retinal sections from Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–hSyn–green fluorescent protein <t>(rAAV-hSyn-GFP;</t> vector control) or rAAV-hSyn-Brn3b virus. The immunostaining was detected using corresponding Alexa 546 (pseudogreen) or Alexa 647 (pseudored) conjugated secondary antibody. Scale bar indicates 20 µm. B : A magnified view of the retinal ganglion cell (RGC) layers of retinas transduced with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. C : A significant 2.4-fold increase in p-AKT expression was observed in the RGCs of rats injected with rAAV-hSyn-Brn3b. Ratios of fluorescence intensity values are shown in mean ± standard error of the mean (SEM), n = 6. The Mann–Whitney rank-sum test was used for statistical analysis (*p
    Gfp Tag Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology rabbit polyclonal anti gfp
    Mitochondrial dynamics in living cells analyzed using <t>GFP</t> photoactivation and time-lapse microscopy. HeLa cells were co-transfected with a mitochondrial matrix targeted photoactivable GFP (mito-PAGFP) and mRFP, or mRFP-Htt28Q or mRFP-Htt74Q expression plasmids. After 24 h transfection, cells were subjected to photoactivation studies, or lysates were prepared from the cells for immunoblot analysis. ( A ) Expression of mRFP, mRFP-Htt28Q and mRFP-Htt74Q proteins detected by immunnoblotting proteins lysates from the transfected cells with a rabbit <t>polyclonal</t> anti-RFP antibody. ( B ) Mitochondria in cells expressing mRFP or mRFP-Htt28Q proteins display more dynamic movement and fusion compared with cells expressing mRFP-Htt74Q protein. HeLa cells that had been co-transfected with mito-PAGFP and mRFP, or mito-PAGFP and mRFP-Htt28Q, or mito-PAGFP and mRFP-Htt74Q were imaged under a fluorescent microscope using a 100× objective lens and those displaying RFP fluorescence were targeted for photoactivation of the co-transfected mito-PAGFP protein by illumination with 405 nm light, and then GFP fluorescence images were captured over the indicated time intervals. Arrowheads point to the mitochondria that fused with one another in the time period shown. Please note that in the GFP-Htt74Q transfected cell, mitochondria were smaller, clustered, lacked dynamic movement and fusion events were very infrequent. ( C ) Same as in (B), except that the images shown were captured using a 40× objective. The left-hand panels show the mRFP fluorescence seen in a group of cells and the region (indicated by the circle) that was illuminated with 405 nm light to photoactivate the co-expressed PAGFP-mito protein in the various transfected cells. The subsequent GFP fluorescence images captured at 0, 15 and 30 min after GFP photoactivation are shown on the right of the RFP fluorescence image captured for each construct. ( D ) Quantification of the changes in GFP fluorescence intensity over time in cells transfected with the different mRFP-tagged expression constructs. GFP fluorescence was measured at 0, 15 and 30 min after photoactivation in the photoactivated (a, b and c) and non-activated regions (e, f and g, respectively) in cells transfected with mRFP or mRFP-Htt28Q or mRFP-Htt74Q expression constructs, respectively. The plots depict the results obtained in 10 independent experiments (each shown with a different color). Note that GFP fluorescence in the photoactivated regions, in general, decreases faster in the cells expressing either mRFP, or mRFP-Htt28Q proteins, compared with those expressing mRFP-Htt74Q protein. These changes were accompanied by a gradual increase in GFP fluorescent intensity in the non-activated regions of cells expressing either mRFP or mRFP-Htt28Q proteins, but remained relatively constant in cells expressing mRFP-Htt74Q. These results are consistent with the idea that mitochondria in cells expressing either the mRFP or the mRFP-HttQ28 proteins exhibit greater mitochondria fusion than cells expressing the mRFP-HttQ74 protein.
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    Image Search Results


    Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.

    Journal: Journal of Virology

    Article Title: Human Immunodeficiency Virus Type 1 Protease Regulation of Tat Activity Is Essential for Efficient Reverse Transcription and Replication

    doi: 10.1128/JVI.77.18.9912-9921.2003

    Figure Lengend Snippet: Cleavage of wild-type (WT) and mutant HIV-1 Tat-GFP proteins by PR. (A) For each PR cleavage assay, 35 S-labeled wild-type and mutant Tat-GFP proteins were synthesized in RRL translation reaction mixtures, and equivalent amounts of the Tat-GFP proteins were mixed as indicated with unlabeled wild-type (plus-strand) or mutant (minus-strand) HIV-1 PR made in separate RRL reaction mixtures. Each experiment was repeated three to six times, and the mean result with standard deviation (error bar) is shown. The level of proteolysis was calculated by comparing the ratio of full-length to cleaved wild-type Tat-GFP protein to the ratio of full-length to cleaved mutant protein. The results were analyzed on a Molecular Dynamics PhosphorImager. (B) Virus stocks were prepared from two independent cell lines making HIV-1Δtat virus and stably transcomplemented with wild-type (WT) or mutant Tat-GFP; the parent cell line is shown as Δtat. The efficiency of minus-strand SS DNA synthesis in HIV-1 made in stably transfected cell lines expressing wild-type or mutated tat was determined by NERT-PCR assays. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. At least three independent virus stocks were collected and assayed, and a representative experiment is shown. The relative fluorescence level of Tat-GFP made by each cell line is shown below the graph. NA, not applicable. (C) Virus stocks collected after transient expression of different Tat-GFP plasmids in 293HIVΔtat cells were assayed by NERT-PCR. The level of minus-strand SS DNA made by virus transcomplemented with wild-type Tat-GFP was set at 100%. These experiments were performed three times, and a representative experiment is shown. (D) Western blot analysis of infected 293 cells stably expressing Tat-GFP using either anti-GFP monoclonal antibody or a purified pooled human anti-HIV-1 polyclonal antibody as indicated.

    Article Snippet: Cell lysate samples were resolved on sodium dodecyl sulfate (SDS)-12.5% polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride and probed with either human anti-HIV-1 immunoglobulin G (IgG) (National Institutes of Health [NIH] AIDS Research and Reference Reagent Program [ARRRP] catalog no. 192; diluted 1:6,000), HIV-1BH10 Tat monoclonal antibody (NIH ARRRP catalog no. 15.1), or anti-GFP rabbit serum (Molecular Probes catalog no. A6455; diluted 1:5,000).

    Techniques: Mutagenesis, Cleavage Assay, Labeling, Synthesized, Standard Deviation, Stable Transfection, DNA Synthesis, Transfection, Expressing, Polymerase Chain Reaction, Fluorescence, Western Blot, Infection, Purification

    The GFAP promoter is astrocyte-specific. (A and B) Two weeks after systemic delivery of either rAAV1/2-GFAP-GFP or rAAV1/2-HBA-GFP, transgene-positive (GFP, green) cells are visible in the FUS-targeted region. Aβ plaque is shown in blue, and GFAP expression in red. (C) At higher magnification in the cortex, the morphology of GFP-positive cells in the rAAV1/2-GFAP-GFP group is consistent and colocalizes with GFAP-positive cells (colocalization, yellow). (D) In the cortex after delivery of rAAV1/2-HBA-GFP, a variety of cell morphologies are visible and are not always colocalized with GFAP. (E and F) In the hippocampus, the same respective trends are seen of GFP-positive cell morphology after delivery of rAAV1/2-GFAP-GFP or rAAV1/2-HBA-GFP. (G) As seen in an orthogonal projection, consistent colocalization between GFP-positive cells and GFAP is verified in the rAAV1/2-GFAP-GFP group. (H) A consistent colocalization between GFP and GFAP is not seen in the orthogonal projection of rAAV1/2-HBA-GFP cells. (I) Quantification of GFP-positive (GFP+) cells are categorized as GFAP+ (astrocyte), or GFAP- (undefined). The percentage of GFP-positive cells that are also GFAP-positive after rAAV1/2-GFAP-GFP delivery indicate almost exclusive transgene expression in astrocytes. (J) The areas containing GFP-positive cells in the cortex and hippocampus after FUS application were not significantly different in size (µm 2 ) between the rAAV1/2-GFAP-GFP and rAAV1/2-HBA-GFP groups (p=0.91). (K) The amount of BBB opening by FUS application can be estimated by the MRI enhancement from background of each focal spot, which was not significantly different between the two rAAV groups (p=0.87). (L) The number of Aβ plaques in the cortical and hippocampal regions containing GFP-positive cells was also not significantly different between rAAV groups (p=0.69). Data is represented as mean ±SEM and (I-L) n=4 animals per group. (K) For MRI enhancement n=12 focal spots from 4 animals, per group. Scale bars: (A and B) 1 mm; (c-f) 100 µm; (G and H) 20 µm.

    Journal: Theranostics

    Article Title: Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease

    doi: 10.7150/thno.36718

    Figure Lengend Snippet: The GFAP promoter is astrocyte-specific. (A and B) Two weeks after systemic delivery of either rAAV1/2-GFAP-GFP or rAAV1/2-HBA-GFP, transgene-positive (GFP, green) cells are visible in the FUS-targeted region. Aβ plaque is shown in blue, and GFAP expression in red. (C) At higher magnification in the cortex, the morphology of GFP-positive cells in the rAAV1/2-GFAP-GFP group is consistent and colocalizes with GFAP-positive cells (colocalization, yellow). (D) In the cortex after delivery of rAAV1/2-HBA-GFP, a variety of cell morphologies are visible and are not always colocalized with GFAP. (E and F) In the hippocampus, the same respective trends are seen of GFP-positive cell morphology after delivery of rAAV1/2-GFAP-GFP or rAAV1/2-HBA-GFP. (G) As seen in an orthogonal projection, consistent colocalization between GFP-positive cells and GFAP is verified in the rAAV1/2-GFAP-GFP group. (H) A consistent colocalization between GFP and GFAP is not seen in the orthogonal projection of rAAV1/2-HBA-GFP cells. (I) Quantification of GFP-positive (GFP+) cells are categorized as GFAP+ (astrocyte), or GFAP- (undefined). The percentage of GFP-positive cells that are also GFAP-positive after rAAV1/2-GFAP-GFP delivery indicate almost exclusive transgene expression in astrocytes. (J) The areas containing GFP-positive cells in the cortex and hippocampus after FUS application were not significantly different in size (µm 2 ) between the rAAV1/2-GFAP-GFP and rAAV1/2-HBA-GFP groups (p=0.91). (K) The amount of BBB opening by FUS application can be estimated by the MRI enhancement from background of each focal spot, which was not significantly different between the two rAAV groups (p=0.87). (L) The number of Aβ plaques in the cortical and hippocampal regions containing GFP-positive cells was also not significantly different between rAAV groups (p=0.69). Data is represented as mean ±SEM and (I-L) n=4 animals per group. (K) For MRI enhancement n=12 focal spots from 4 animals, per group. Scale bars: (A and B) 1 mm; (c-f) 100 µm; (G and H) 20 µm.

    Article Snippet: The primary antibodies used were the same rabbit anti-GFP (1:500), donkey anti-GFAP (1:300), and DAPI as described above.

    Techniques: Expressing, Magnetic Resonance Imaging

    GFAP promoter permits transgene expression in the liver. (A) Under control of the GFAP promoter, GFP expression (green) was not prevented in the liver, despite an absence of GFAP protein detection (red). Cell nuclei are shown in blue (DAPI). (B) Under control of the HBA promoter, GFP was also expressed in the liver. (C and D) Both promoters lead to expression in the kidney, (E and F) while only the rAAV1/2-HBA-GFP group showed GFP expression in the heart. (G) GFP expression was not seen in the quadriceps muscle of the rAAV1/2-GFAP-GFP group, (H) but was detected in the rAAV1/2-HBA-GFP group. (I) GFP expression was not detected in the spleen after delivery of rAAV1/2-GFAP-GFP, (J) but was detected in the rAAV1/2-HBA-GFP group. (K and L) No GFP expression was detected in the lung for either the GFAP or HBA promoter groups. (A-L) Scale bar, 50 µm.

    Journal: Theranostics

    Article Title: Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease

    doi: 10.7150/thno.36718

    Figure Lengend Snippet: GFAP promoter permits transgene expression in the liver. (A) Under control of the GFAP promoter, GFP expression (green) was not prevented in the liver, despite an absence of GFAP protein detection (red). Cell nuclei are shown in blue (DAPI). (B) Under control of the HBA promoter, GFP was also expressed in the liver. (C and D) Both promoters lead to expression in the kidney, (E and F) while only the rAAV1/2-HBA-GFP group showed GFP expression in the heart. (G) GFP expression was not seen in the quadriceps muscle of the rAAV1/2-GFAP-GFP group, (H) but was detected in the rAAV1/2-HBA-GFP group. (I) GFP expression was not detected in the spleen after delivery of rAAV1/2-GFAP-GFP, (J) but was detected in the rAAV1/2-HBA-GFP group. (K and L) No GFP expression was detected in the lung for either the GFAP or HBA promoter groups. (A-L) Scale bar, 50 µm.

    Article Snippet: The primary antibodies used were the same rabbit anti-GFP (1:500), donkey anti-GFAP (1:300), and DAPI as described above.

    Techniques: Expressing

    GFAP promoter-driven expression of GFP intensified in the vicinity of Aβ plaque. (A-D) rAAV1/2-GFAP-GFP expression in GFAP-positive cells (red) with processes overlapping in space with Aβ plaque (blue) show a distinct pattern of expression in both the cell body and processes compared to (E-H) rAAV1/2-GFAP-GFP expression in the absence of Aβ plaque and rAAV1/2-HBA-GFP expression both (I-L) associated and (M-P) unassociated with Aβ plaque. Scale bar, 20 µm.

    Journal: Theranostics

    Article Title: Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease

    doi: 10.7150/thno.36718

    Figure Lengend Snippet: GFAP promoter-driven expression of GFP intensified in the vicinity of Aβ plaque. (A-D) rAAV1/2-GFAP-GFP expression in GFAP-positive cells (red) with processes overlapping in space with Aβ plaque (blue) show a distinct pattern of expression in both the cell body and processes compared to (E-H) rAAV1/2-GFAP-GFP expression in the absence of Aβ plaque and rAAV1/2-HBA-GFP expression both (I-L) associated and (M-P) unassociated with Aβ plaque. Scale bar, 20 µm.

    Article Snippet: The primary antibodies used were the same rabbit anti-GFP (1:500), donkey anti-GFAP (1:300), and DAPI as described above.

    Techniques: Expressing

    GFAP promoter results in greater GFP fluorescence intensity, volume, and surface area of astrocytes near Aβ plaque. (A) Quantification of GFP fluorescence per unit volume is significantly increased in GFAP and GFP-positive cells near Aβ plaque in the rAAV1/2-GFAP-GFP group, compared to GFAP and GFP-positive cells unassociated with Aβ plaque (**p

    Journal: Theranostics

    Article Title: Strategy to enhance transgene expression in proximity of amyloid plaques in a mouse model of Alzheimer's disease

    doi: 10.7150/thno.36718

    Figure Lengend Snippet: GFAP promoter results in greater GFP fluorescence intensity, volume, and surface area of astrocytes near Aβ plaque. (A) Quantification of GFP fluorescence per unit volume is significantly increased in GFAP and GFP-positive cells near Aβ plaque in the rAAV1/2-GFAP-GFP group, compared to GFAP and GFP-positive cells unassociated with Aβ plaque (**p

    Article Snippet: The primary antibodies used were the same rabbit anti-GFP (1:500), donkey anti-GFAP (1:300), and DAPI as described above.

    Techniques: Fluorescence

    The crosstalk between shrimp p53 and NF-κB pathways. ( A , B ) The interaction between LvFLp53/LvΔNp53 and LvDorsal. Co-immunoprecipitation assays showed that the GFP-tagged LvDorsal but not the control GFP protein could be co-precipitated by FLAG-tagged LvFLp53 ( A ) and FLAG-tagged LvΔNp53 ( B ), respectively. Immunoprecipitation (IP) and western-blotting are performed using anti-V5 and anti-GFP antibodies, respectively. Approximate molecular sizes: LvFLp53-FLAG, ~54 kDa; LvΔNp53-FLAG, ~38 kDa; LvDorsal-GFP, ~72 kDa; GFP, ~28 kDa. ( C ) The effects of LvFLp53 and LvΔNp53 on the activity of NF-κB (LvDorsal). Drosophila S2 cells are co-transfected with pAc5.1-LvDorsal and an increasing amount of pAc5.1-LvFLp53 and/or pAc5.1-LvΔNp53, and the effects on the promoter containing four NF-κB binding domains were detected by dual luciferase reporter assays. ( D ) The effects of LvDorsal or LvDorsal co-expressed with LvFLp53 or/and LvΔNp53 on the promoters activities of WSSV IE genes. Drosophila S2 cells are co-transfected with pAc5.1-LvDorsal or/and pAc5.1-LvFLp53 and/or pAc5.1-LvΔNp53, and the effects were detected by dual luciferase reporter assays. ( E ) A possible regulatory mechanism of LvFLp53 and LvΔNp53 in NF-κB mediated immune response (See in details in discussion). All results are representative of three independent experiments.

    Journal: Scientific Reports

    Article Title: Identification of two p53 isoforms from Litopenaeus vannamei and their interaction with NF-κB to induce distinct immune response

    doi: 10.1038/srep45821

    Figure Lengend Snippet: The crosstalk between shrimp p53 and NF-κB pathways. ( A , B ) The interaction between LvFLp53/LvΔNp53 and LvDorsal. Co-immunoprecipitation assays showed that the GFP-tagged LvDorsal but not the control GFP protein could be co-precipitated by FLAG-tagged LvFLp53 ( A ) and FLAG-tagged LvΔNp53 ( B ), respectively. Immunoprecipitation (IP) and western-blotting are performed using anti-V5 and anti-GFP antibodies, respectively. Approximate molecular sizes: LvFLp53-FLAG, ~54 kDa; LvΔNp53-FLAG, ~38 kDa; LvDorsal-GFP, ~72 kDa; GFP, ~28 kDa. ( C ) The effects of LvFLp53 and LvΔNp53 on the activity of NF-κB (LvDorsal). Drosophila S2 cells are co-transfected with pAc5.1-LvDorsal and an increasing amount of pAc5.1-LvFLp53 and/or pAc5.1-LvΔNp53, and the effects on the promoter containing four NF-κB binding domains were detected by dual luciferase reporter assays. ( D ) The effects of LvDorsal or LvDorsal co-expressed with LvFLp53 or/and LvΔNp53 on the promoters activities of WSSV IE genes. Drosophila S2 cells are co-transfected with pAc5.1-LvDorsal or/and pAc5.1-LvFLp53 and/or pAc5.1-LvΔNp53, and the effects were detected by dual luciferase reporter assays. ( E ) A possible regulatory mechanism of LvFLp53 and LvΔNp53 in NF-κB mediated immune response (See in details in discussion). All results are representative of three independent experiments.

    Article Snippet: Western blotting was performed with rabbit anti-GFP antibody (Sigma, USA) and rabbit anti-FLAG antibody (Sigma, USA), and alkaline phosphatase-conjugated goat anti-rabbit secondary antibodies (Sigma, USA).

    Techniques: Immunoprecipitation, Western Blot, Activity Assay, Transfection, Binding Assay, Luciferase

    Distribution of Src1 at the nuclear envelope. ( A ) Localization of GFP-Src1 during interphase and mitosis. Mitotic stages are indicated. GFP fluorescence is shown in green, DAPI staining of nuclei in blue, and microtubules in red (labeled with anti-α-tubulin YL1/2 and anti-rat-AlexaFluor 561). Bar, 4 μm. ( B ) Localization of endogenous Src1 labeled with anti-Src1/anti-rabbit AlexaFluor 488 (green), anti-NE81/anti-AlexaFluor 568 (red), and DAPI (blue). Note the uneven distribution of Src1 with accumulations at sites of nucleolar attachment to the nuclear envelope. Nucleoli (arrowheads) are poorly stained with DAPI and visible in phase contrast images as dark structures within the nucleus. ( A, B ) widefield deconvolution microscopy. Specimens were fixed with glutaraldehyde. Maximum intensity projections of thin image stacks through the center of the nucleus are shown. Bar, 5 μm. ( B' ) Quantitative evaluation of anti-Src1 staining intensities in nucleolar (No) vs. non-nucleolar (non-No) regions at the NE. Mean normalized staining intensities ± S.D. are shown ( n = 60 for each region). ( C ) Western blot of cytosolic extracts of wild-type, GFP-Src1 full length, and GFP-Src1 1–646 cells stained with anti-Src1 antibodies and anti-GFP antibodies as indicated. Numbers refer to relative staining intensities, whereby Src1 staining in wild-type cells was set to 1. As protein levels of GFP-Src1 are the same whether stained with anti-Src1 or anti-GFP, the protein level of GFP- Src1 1–646 compared to endogenous Src1 could be deduced. Actin is shown as a loading control.

    Journal: Cells

    Article Title: Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    doi: 10.3390/cells5010013

    Figure Lengend Snippet: Distribution of Src1 at the nuclear envelope. ( A ) Localization of GFP-Src1 during interphase and mitosis. Mitotic stages are indicated. GFP fluorescence is shown in green, DAPI staining of nuclei in blue, and microtubules in red (labeled with anti-α-tubulin YL1/2 and anti-rat-AlexaFluor 561). Bar, 4 μm. ( B ) Localization of endogenous Src1 labeled with anti-Src1/anti-rabbit AlexaFluor 488 (green), anti-NE81/anti-AlexaFluor 568 (red), and DAPI (blue). Note the uneven distribution of Src1 with accumulations at sites of nucleolar attachment to the nuclear envelope. Nucleoli (arrowheads) are poorly stained with DAPI and visible in phase contrast images as dark structures within the nucleus. ( A, B ) widefield deconvolution microscopy. Specimens were fixed with glutaraldehyde. Maximum intensity projections of thin image stacks through the center of the nucleus are shown. Bar, 5 μm. ( B' ) Quantitative evaluation of anti-Src1 staining intensities in nucleolar (No) vs. non-nucleolar (non-No) regions at the NE. Mean normalized staining intensities ± S.D. are shown ( n = 60 for each region). ( C ) Western blot of cytosolic extracts of wild-type, GFP-Src1 full length, and GFP-Src1 1–646 cells stained with anti-Src1 antibodies and anti-GFP antibodies as indicated. Numbers refer to relative staining intensities, whereby Src1 staining in wild-type cells was set to 1. As protein levels of GFP-Src1 are the same whether stained with anti-Src1 or anti-GFP, the protein level of GFP- Src1 1–646 compared to endogenous Src1 could be deduced. Actin is shown as a loading control.

    Article Snippet: Antibodies and Streptavidin Conjugates Primary antibodies: rat anti-Src1 (this work), rabbit anti-NE81 [ ], rat YL1/2 [ ], rabbit anti-GFP [ ], rabbit anti-GFP (Molecular Probes, A-6455; Life Technologies, Carlsbad, CA, USA).

    Techniques: Fluorescence, Staining, Labeling, Microscopy, Western Blot

    Nuclear envelope protrusions caused by expression of GFP-Src1 1–646 are dependent on intact microtubules but not actin filaments. ( A ) Schematic of GFP-Src1 1–646 . ( B ) Western Blot stained with anti-GFP antibodies showing GFP-Src1 1–646 membrane association after extractions with buffer (control), high-salt, and detergent, respectively. GFP-Src1 1–646 is a nuclear membrane protein, since it becomes solubilized only by extraction with 1% Triton-X100. ( C ) Immunofluorescence microscopy of paraformaldehyde fixed GFP-Src1 1–646 (green) cells stained with anti-NE81/AlexaFluor 568 (red) and DAPI (blue). Bar, 5 μm. Note that the origin of the nuclear envelope protrusion (arrowhead) is associated with a nucleolus. ( D – E ) Selected time points of supplemental movie 4 ( D ) and movie 5 ( E ) showing the dynamic behavior of nuclear membrane protrusions (arrowhead). GFP-Src1 1–646 cells (hash tag) were mixed 1:1 either with GFP-LIMΔcoil cells(asterisk) [ 45 ] with green fluorescent F-actin ( D ) or with GFP-α-tubulin cells (asterisk) [ 46 ] with green fluorescent microtubules ( E ) to monitor effectiveness of drug treatments. The presence of protusions was not affected by treatment with 200 μM latrunculin A (LatA, D' ) or 100 μM thiabendazole (TBZ, E' ). Cells were viewed under agar overlay. Bars, 10 μm.

    Journal: Cells

    Article Title: Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    doi: 10.3390/cells5010013

    Figure Lengend Snippet: Nuclear envelope protrusions caused by expression of GFP-Src1 1–646 are dependent on intact microtubules but not actin filaments. ( A ) Schematic of GFP-Src1 1–646 . ( B ) Western Blot stained with anti-GFP antibodies showing GFP-Src1 1–646 membrane association after extractions with buffer (control), high-salt, and detergent, respectively. GFP-Src1 1–646 is a nuclear membrane protein, since it becomes solubilized only by extraction with 1% Triton-X100. ( C ) Immunofluorescence microscopy of paraformaldehyde fixed GFP-Src1 1–646 (green) cells stained with anti-NE81/AlexaFluor 568 (red) and DAPI (blue). Bar, 5 μm. Note that the origin of the nuclear envelope protrusion (arrowhead) is associated with a nucleolus. ( D – E ) Selected time points of supplemental movie 4 ( D ) and movie 5 ( E ) showing the dynamic behavior of nuclear membrane protrusions (arrowhead). GFP-Src1 1–646 cells (hash tag) were mixed 1:1 either with GFP-LIMΔcoil cells(asterisk) [ 45 ] with green fluorescent F-actin ( D ) or with GFP-α-tubulin cells (asterisk) [ 46 ] with green fluorescent microtubules ( E ) to monitor effectiveness of drug treatments. The presence of protusions was not affected by treatment with 200 μM latrunculin A (LatA, D' ) or 100 μM thiabendazole (TBZ, E' ). Cells were viewed under agar overlay. Bars, 10 μm.

    Article Snippet: Antibodies and Streptavidin Conjugates Primary antibodies: rat anti-Src1 (this work), rabbit anti-NE81 [ ], rat YL1/2 [ ], rabbit anti-GFP [ ], rabbit anti-GFP (Molecular Probes, A-6455; Life Technologies, Carlsbad, CA, USA).

    Techniques: Expressing, Western Blot, Staining, Immunofluorescence, Microscopy

    GFP-Src1 exhibits low mobility in FRAP experiments. ( A ) Selected time points of supplemental movie 2 are shown. The bleached region is indicated by a white square. Bar, 5 μm. ( B ) FRAP curve showing mean values ± S.D. after normalization and background correction ( n = 8).

    Journal: Cells

    Article Title: Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    doi: 10.3390/cells5010013

    Figure Lengend Snippet: GFP-Src1 exhibits low mobility in FRAP experiments. ( A ) Selected time points of supplemental movie 2 are shown. The bleached region is indicated by a white square. Bar, 5 μm. ( B ) FRAP curve showing mean values ± S.D. after normalization and background correction ( n = 8).

    Article Snippet: Antibodies and Streptavidin Conjugates Primary antibodies: rat anti-Src1 (this work), rabbit anti-NE81 [ ], rat YL1/2 [ ], rabbit anti-GFP [ ], rabbit anti-GFP (Molecular Probes, A-6455; Life Technologies, Carlsbad, CA, USA).

    Techniques:

    mRFP-Src1 356–565 and mRFP-Src1 826–942 localize to cytosolic GFP-NE81ΔNLSΔCLIM clusters. ( A ) Selected time points of supplemental movie 3 showing the dynamic behavior of GFP-NE81ΔNLSΔCLIM clusters. ( B, C ) Transmission electron microscopy showing spongy GFP-NE81ΔNLSΔCLIM clusters (Cl) studded by particles representing ribosomes. The nucleus (Nu), nucleoli (No) and mitochondria (Mi) are labeled. ( C ) is an enlarged view of ( B ). ( D ) Schematic of mRFP-Src1 fragments used in ( E–H ). ( E, F ) mRFP-Src1 356–565 and mRFP-Src1 826–942 (red) predominantly localize to the nucleus (stained with DAPI, blue). mRFP-Src1 826–942 is concentrated at regions with low DAPI staining indicating presence at the nucleoli. Cells were fixed with glutaraldehyde. ( G, H ) In live GFP-NE81ΔNLSΔCLIM cells mRFP-Src1 356–565 and mRFP-Src1 826–942 (red) mainly localize to GFP-NE81ΔNLSΔCLIM clusters. Bars, 5 μm.

    Journal: Cells

    Article Title: Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    doi: 10.3390/cells5010013

    Figure Lengend Snippet: mRFP-Src1 356–565 and mRFP-Src1 826–942 localize to cytosolic GFP-NE81ΔNLSΔCLIM clusters. ( A ) Selected time points of supplemental movie 3 showing the dynamic behavior of GFP-NE81ΔNLSΔCLIM clusters. ( B, C ) Transmission electron microscopy showing spongy GFP-NE81ΔNLSΔCLIM clusters (Cl) studded by particles representing ribosomes. The nucleus (Nu), nucleoli (No) and mitochondria (Mi) are labeled. ( C ) is an enlarged view of ( B ). ( D ) Schematic of mRFP-Src1 fragments used in ( E–H ). ( E, F ) mRFP-Src1 356–565 and mRFP-Src1 826–942 (red) predominantly localize to the nucleus (stained with DAPI, blue). mRFP-Src1 826–942 is concentrated at regions with low DAPI staining indicating presence at the nucleoli. Cells were fixed with glutaraldehyde. ( G, H ) In live GFP-NE81ΔNLSΔCLIM cells mRFP-Src1 356–565 and mRFP-Src1 826–942 (red) mainly localize to GFP-NE81ΔNLSΔCLIM clusters. Bars, 5 μm.

    Article Snippet: Antibodies and Streptavidin Conjugates Primary antibodies: rat anti-Src1 (this work), rabbit anti-NE81 [ ], rat YL1/2 [ ], rabbit anti-GFP [ ], rabbit anti-GFP (Molecular Probes, A-6455; Life Technologies, Carlsbad, CA, USA).

    Techniques: Transmission Assay, Electron Microscopy, Labeling, Staining

    Src1 is an inner nuclear membrane protein. ( A, B ) Isolated nuclei of GFP-Src1 cells were stained with anti-GFP-antibodies [ 33 ] either in the absence ( A ) or presence ( B ) of 0.5% Triton-X100. The antibody is accessible to GFP-Src1 only upon permeabilization of the nuclear membranes. Specimens were fixed with glutaraldehyde. Bar, 2 μm. ( C, C' ) Two examples of immuno-transmission electron microscopy of isolated nuclei stained with anti-GFP/anti-rabbit nanogold. Gold particles (arrowheads) are visible only along the inner nuclear membrane. Bar, 500 nm.

    Journal: Cells

    Article Title: Src1 is a Protein of the Inner Nuclear Membrane Interacting with the Dictyostelium Lamin NE81

    doi: 10.3390/cells5010013

    Figure Lengend Snippet: Src1 is an inner nuclear membrane protein. ( A, B ) Isolated nuclei of GFP-Src1 cells were stained with anti-GFP-antibodies [ 33 ] either in the absence ( A ) or presence ( B ) of 0.5% Triton-X100. The antibody is accessible to GFP-Src1 only upon permeabilization of the nuclear membranes. Specimens were fixed with glutaraldehyde. Bar, 2 μm. ( C, C' ) Two examples of immuno-transmission electron microscopy of isolated nuclei stained with anti-GFP/anti-rabbit nanogold. Gold particles (arrowheads) are visible only along the inner nuclear membrane. Bar, 500 nm.

    Article Snippet: Antibodies and Streptavidin Conjugates Primary antibodies: rat anti-Src1 (this work), rabbit anti-NE81 [ ], rat YL1/2 [ ], rabbit anti-GFP [ ], rabbit anti-GFP (Molecular Probes, A-6455; Life Technologies, Carlsbad, CA, USA).

    Techniques: Isolation, Staining, Transmission Assay, Electron Microscopy

    BCAAem recruits endothelial progenitors to dystrophic VM muscles. Cryosections of VM ( a ) muscle tissues of BCAAem-treated and untreated C57BL6/J- EGFP and mdx-EGFP mice were were immunostained for GFP, and costained for CD31 and laminin (lam) (n = 5 for each experimental group). Nuclei were stained with DAPI. EGFP+ cells were detected in the interstitial spaces (arrowheads in a) and inside small CD31+ vessels (boxes in a). Magnification (X 1,000) of boxes indicate vessels expressing GFP (green), laminin (red), CD31 (purple). Merged images reveal vessel areas of costaining, as evidenced by the grey staining pattern. Scale bar, 45 µm. Quantifications of EGFP+ cells ( b ) and EGFP+ CD31+ cells ( c ) in each section and CD31+ vessels per fibre ( d ) and α-SMA+ cells per section ( e ) are shown. All data are presented as the mean ± s.e.m. Statistical error analysis was performed by one-way ANOVA with Bonferroni correction; **p

    Journal: Scientific Reports

    Article Title: Supplementation with a selective amino acid formula ameliorates muscular dystrophy in mdx mice

    doi: 10.1038/s41598-018-32613-w

    Figure Lengend Snippet: BCAAem recruits endothelial progenitors to dystrophic VM muscles. Cryosections of VM ( a ) muscle tissues of BCAAem-treated and untreated C57BL6/J- EGFP and mdx-EGFP mice were were immunostained for GFP, and costained for CD31 and laminin (lam) (n = 5 for each experimental group). Nuclei were stained with DAPI. EGFP+ cells were detected in the interstitial spaces (arrowheads in a) and inside small CD31+ vessels (boxes in a). Magnification (X 1,000) of boxes indicate vessels expressing GFP (green), laminin (red), CD31 (purple). Merged images reveal vessel areas of costaining, as evidenced by the grey staining pattern. Scale bar, 45 µm. Quantifications of EGFP+ cells ( b ) and EGFP+ CD31+ cells ( c ) in each section and CD31+ vessels per fibre ( d ) and α-SMA+ cells per section ( e ) are shown. All data are presented as the mean ± s.e.m. Statistical error analysis was performed by one-way ANOVA with Bonferroni correction; **p

    Article Snippet: To detect EGFP+ cells, slides were incubated with rabbit anti-GFP antibody (1:100; Molecular Probes, Thermo Scientific, USA).

    Techniques: Mouse Assay, Laser Capture Microdissection, Staining, Expressing

    BCAAem recruits endothelial progenitors to healthy and dystrophic TA muscles. Cryosections of TA ( a ) muscle tissues of BCAAem-treated and untreated C57BL6/J- EGFP and mdx-EGFP mice were analysed by immunofluorescence staining for GFP, CD31 and laminin (Lam) (n = 5 for each experimental group). Nuclei were stained with DAPI. EGFP+ cells were detected in the interstitial spaces (arrowheads in a) and inside small CD31+ vessels (boxes in a). Magnification (X 1,000) of boxes indicate vessels expressing GFP (green), laminin (red), CD31 (purple). Merged images reveal vessel areas of costaining, as evidenced by the grey staining pattern. Analysis of the TA muscles of BCAAem-treated mdx-EGFP mice demonstrates an increase in the number of EGFP+ CD31+ and CD31+ vessels and α-SMA+ cells per section. Scale bar, 45 µm. The percentage of EGFP+ cells per section ( b ) and the number of EGFP+ CD31+ cells per section ( c ) are shown. The number of CD31+ vessels per fibre ( d ) and α-SMA+ cells per section ( e ) is shown. All data are presented as the mean ± s.e.m. Statistical error analysis was performed by one-way ANOVA with Bonferroni correction; *p

    Journal: Scientific Reports

    Article Title: Supplementation with a selective amino acid formula ameliorates muscular dystrophy in mdx mice

    doi: 10.1038/s41598-018-32613-w

    Figure Lengend Snippet: BCAAem recruits endothelial progenitors to healthy and dystrophic TA muscles. Cryosections of TA ( a ) muscle tissues of BCAAem-treated and untreated C57BL6/J- EGFP and mdx-EGFP mice were analysed by immunofluorescence staining for GFP, CD31 and laminin (Lam) (n = 5 for each experimental group). Nuclei were stained with DAPI. EGFP+ cells were detected in the interstitial spaces (arrowheads in a) and inside small CD31+ vessels (boxes in a). Magnification (X 1,000) of boxes indicate vessels expressing GFP (green), laminin (red), CD31 (purple). Merged images reveal vessel areas of costaining, as evidenced by the grey staining pattern. Analysis of the TA muscles of BCAAem-treated mdx-EGFP mice demonstrates an increase in the number of EGFP+ CD31+ and CD31+ vessels and α-SMA+ cells per section. Scale bar, 45 µm. The percentage of EGFP+ cells per section ( b ) and the number of EGFP+ CD31+ cells per section ( c ) are shown. The number of CD31+ vessels per fibre ( d ) and α-SMA+ cells per section ( e ) is shown. All data are presented as the mean ± s.e.m. Statistical error analysis was performed by one-way ANOVA with Bonferroni correction; *p

    Article Snippet: To detect EGFP+ cells, slides were incubated with rabbit anti-GFP antibody (1:100; Molecular Probes, Thermo Scientific, USA).

    Techniques: Mouse Assay, Immunofluorescence, Staining, Laser Capture Microdissection, Expressing

    The SNMP1 ectodomain, but not the transmembrane or intracellular domains, is required for pheromone detection. ( a – d ) Analysis of SNMP1 ΔN-term , SNMP1 ΔC-term , NINAD:EGFP (for immunohistochemistry) or untagged NINAD (for electrophysiology) and SNMP1/NINAD:GFP chimera rescue transgenes. Left: immunostaining with α-SNMP1 ( a , b ) or α-GFP ( c , d ) on antennal cryosections. Scale bars, 20 μm. As SNMP1 ΔC-term lacks the C-terminal peptide epitope of the SNMP1 antibody 16 , we used an antibody raised against an SNMP1 ectodomain peptide; although this antibody recognizes SNMP1 in soma, it does not label cilia-localized SNMP1, even in wild-type flies, precluding direct visualization of SNMP1 ΔC-term in this sensory compartment. Centre: representative traces of extracellular electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P

    Journal: Nature Communications

    Article Title: A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism

    doi: 10.1038/ncomms11866

    Figure Lengend Snippet: The SNMP1 ectodomain, but not the transmembrane or intracellular domains, is required for pheromone detection. ( a – d ) Analysis of SNMP1 ΔN-term , SNMP1 ΔC-term , NINAD:EGFP (for immunohistochemistry) or untagged NINAD (for electrophysiology) and SNMP1/NINAD:GFP chimera rescue transgenes. Left: immunostaining with α-SNMP1 ( a , b ) or α-GFP ( c , d ) on antennal cryosections. Scale bars, 20 μm. As SNMP1 ΔC-term lacks the C-terminal peptide epitope of the SNMP1 antibody 16 , we used an antibody raised against an SNMP1 ectodomain peptide; although this antibody recognizes SNMP1 in soma, it does not label cilia-localized SNMP1, even in wild-type flies, precluding direct visualization of SNMP1 ΔC-term in this sensory compartment. Centre: representative traces of extracellular electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. in each genotype. There are significant statistical differences in neuronal responses due to genotype for both 1 and 10% cVA (Kruskal–Wallis, P

    Article Snippet: The primary antibodies used were rabbit α-SNMP1 (C terminus) (diluted 1:200), rabbit α-GFP (Invitrogen A-6455) (1:1,000) and guinea pig α-ORCO (1:1,000).

    Techniques: Immunohistochemistry, Immunostaining

    Evolutionary conservation of SNMP1 function. ( a ) Analysis of snmp1 rescue flies ( Or67d-GAL4/UAS-XXX;snmp1 1 /snmp1 2 , in this and all equivalent rescue experiments) expressing C-terminal EGFP fusions of SNMP1 from D. melanogaster , A. polyphemus and A. mellifera . Left: immunostaining with α-GFP on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. to the indicated stimuli in each genotype. There are significant statistical differences in neuronal responses to cVA due to genotype (Kruskal–Wallis, P

    Journal: Nature Communications

    Article Title: A CD36 ectodomain mediates insect pheromone detection via a putative tunnelling mechanism

    doi: 10.1038/ncomms11866

    Figure Lengend Snippet: Evolutionary conservation of SNMP1 function. ( a ) Analysis of snmp1 rescue flies ( Or67d-GAL4/UAS-XXX;snmp1 1 /snmp1 2 , in this and all equivalent rescue experiments) expressing C-terminal EGFP fusions of SNMP1 from D. melanogaster , A. polyphemus and A. mellifera . Left: immunostaining with α-GFP on antennal cryosections. Scale bars, 20 μm. Centre: representative traces of electrophysiological recordings of OR67d neurons in male flies stimulated with 10% cVA. Right: mean neuronal responses±s.e.m. to the indicated stimuli in each genotype. There are significant statistical differences in neuronal responses to cVA due to genotype (Kruskal–Wallis, P

    Article Snippet: The primary antibodies used were rabbit α-SNMP1 (C terminus) (diluted 1:200), rabbit α-GFP (Invitrogen A-6455) (1:1,000) and guinea pig α-ORCO (1:1,000).

    Techniques: Expressing, Immunostaining

    RABGEF1 is recruited to the damaged mitochondria in a ubiquitin-binding dependent manner. ( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding.

    Journal: eLife

    Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

    doi: 10.7554/eLife.31326

    Figure Lengend Snippet: RABGEF1 is recruited to the damaged mitochondria in a ubiquitin-binding dependent manner. ( A ) HeLa cells transiently expressing mChery-Parkin and GFP-mRABGEF1 were treated with DMSO or valinomycin for 3 hr followed by immunostaining. The magnified pictures were shown in the right. Bars, 10 μm. ( B ) Total cell lysates of ( A ) were analyzed by immunoblotting. Anti-GFP antibody was used for the GFP-mRABGEF1 detection. * and # denote ubiquitinated forms and truncated forms, respectively. ( C ) Quantification of RABGEF1 recruitment to damaged mitochondria in ( A ). None, partial and complete denote that GFP-mRABGEF1 signals were overlapped with no, some of, and all mitochondria, respectively. ( D ) Recombinant ubiquitin (Ub) pre-treated with or without GST-TcPINK1 was subjected to pull-down assay with GST-mRABGEF1. W and E indicate wash and eluted fractions, respectively. 10%, 10% of input. ( E ) Percentages of the amount of ubiquitin in the eluted fraction in ( D ) were shown. The error bars represent mean ±SE from three independent experiments. ( F ) K48-linked and K63-linked Ub chains pre-treated with or without GST-TcPINK1 were subjected to pull-down assay with GST-mRABGEF1. ( G ) Interactions between GST-mRABGEF1 (WT or Y26A/A58D) and ubiquitin or phosphorylated ubiquitin were measured by ITC. N, stoichiometry of binding.

    Article Snippet: The following antibodies were used for immunostaining: rabbit anti-GFP (A-11122; Invitrogen, Grand Island, NY), mouse anti-GFP (A-11120; Invitrogen), rabbit anti-GFP (ab6556; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology), mouse anti-TOMM20 (sc-17764 Clone F-10; Santa Cruz Biotechnology), mouse anti-HA (M180-3; MBL Life science, Japan), mouse anti-HA (HA.11 Clone 16B12; COVANCE, Berkeley, CA), mouse anti-LAMP2 (sc-18822; Santa Cruz Biotechnology), mouse anti-EEA1 (610457; BD Biosciences, San Jose, CA), mouse anti-GM130 (610822; BD Biosciences), mouse anti-pryruvate dehydrogenase E1-alpha subunit (PDHA1) (ab110334; Abcam), mouse anti-DNA (CBL186; Millipore), rabbit anti-RAB5 (C8B1; Cell Signaling Technology, Beverly, MA), rabbit anti-RAB7 (D95F2; Cell Signaling Technology), purified rabbit anti-TBC1D15 (a kind gift from N. Ishihara), and rabbit anti-ATG9A and anti-ATG16L1 (kind gifts from N. Mizushima).

    Techniques: Binding Assay, Expressing, Immunostaining, Recombinant, Pull Down Assay

    RAB7A directly associates to the outer membrane of damaged mitochondria. ( A and B ) The indicated HCT116 cells stably expressing YFP-LC3B, mCherry-Parkin and 2HA-RAB7A were treated with DMSO ( A ) or valinomycin ( B ) for 3 hr, and subjected to immunostaining. The magnified images of the cells treated with valinomycin were shown in a-f. Bars, 10 μm. ( C ) TBC1D15/17 DKO cells stably expressing mCherry-Parkin and YFP-RAB7A were treated with DMSO ( a and b ) or valinomycin ( c–f ) for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. Panels b and d are the magnified images of boxes in panels a and c, respectively. Bars, 500 nm.

    Journal: eLife

    Article Title: Endosomal Rab cycles regulate Parkin-mediated mitophagy

    doi: 10.7554/eLife.31326

    Figure Lengend Snippet: RAB7A directly associates to the outer membrane of damaged mitochondria. ( A and B ) The indicated HCT116 cells stably expressing YFP-LC3B, mCherry-Parkin and 2HA-RAB7A were treated with DMSO ( A ) or valinomycin ( B ) for 3 hr, and subjected to immunostaining. The magnified images of the cells treated with valinomycin were shown in a-f. Bars, 10 μm. ( C ) TBC1D15/17 DKO cells stably expressing mCherry-Parkin and YFP-RAB7A were treated with DMSO ( a and b ) or valinomycin ( c–f ) for 3 hr and then subjected to immunoelectron microscopy with anti-GFP antibody. Panels b and d are the magnified images of boxes in panels a and c, respectively. Bars, 500 nm.

    Article Snippet: The following antibodies were used for immunostaining: rabbit anti-GFP (A-11122; Invitrogen, Grand Island, NY), mouse anti-GFP (A-11120; Invitrogen), rabbit anti-GFP (ab6556; Abcam), rabbit anti-TOMM20 (sc-11415; Santa Cruz Biotechnology), mouse anti-TOMM20 (sc-17764 Clone F-10; Santa Cruz Biotechnology), mouse anti-HA (M180-3; MBL Life science, Japan), mouse anti-HA (HA.11 Clone 16B12; COVANCE, Berkeley, CA), mouse anti-LAMP2 (sc-18822; Santa Cruz Biotechnology), mouse anti-EEA1 (610457; BD Biosciences, San Jose, CA), mouse anti-GM130 (610822; BD Biosciences), mouse anti-pryruvate dehydrogenase E1-alpha subunit (PDHA1) (ab110334; Abcam), mouse anti-DNA (CBL186; Millipore), rabbit anti-RAB5 (C8B1; Cell Signaling Technology, Beverly, MA), rabbit anti-RAB7 (D95F2; Cell Signaling Technology), purified rabbit anti-TBC1D15 (a kind gift from N. Ishihara), and rabbit anti-ATG9A and anti-ATG16L1 (kind gifts from N. Mizushima).

    Techniques: Stable Transfection, Expressing, Immunostaining, Immuno-Electron Microscopy

    RAB7L1 causes recruitment of LRRK2 in a manner independent of kinase activity. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 (green), or cotransfected with wildtype LRRK2 and wildtype or mutant mRFP-tagged RAB7L1 (red) as indicated, and stained with DAPI (blue). Scale bar, 10 μm. (B) Example of cells cotransfected with the indicated pathogenic GFP-LRRK2 mutants (green) and mRFP-RAB7L1 (red) as indicated, and stained with DAPI (blue). Scale bar, 10 μm. (C) Example of cells cotransfected with mRFP-RAB7L1 (red) and either wildtype or K1906M kinase-dead mutant GFP-LRRK2 (green), or cotransfected with wildtype GFP-LRRK2 (green) and treated with 500 nM LRRK2-IN-1 (INH1) or 500 nM GSK2578215A (GSK) for 60 min before fixation, and stained with DAPI (blue). Scale bar, 10 μm. (D) Quantification of the percentage of colocalization of the indicated LRRK2 constructs with RAB7L1 as determined by analysis of Mander’s coefficient (M2 × 100) from 10 random cells per condition. (E) Cells were cotransfected with the indicated GFP-tagged LRRK2 constructs and flag-tagged RAB7L1, and treated with 100 nM MLi2 for 60 min where indicated. Binding between LRRK2 and RAB7L1 was assessed by co-immunoprecipitation of GFP-tagged LRRK2 variants with flag-tagged RAB7L1 using a polyclonal GFP antibody. Left panel shows inputs, and right panel shows samples after immunoprecipitation, probed either for GFP (using a monoclonal GFP antibody), or in a separate gel probed for flag and tubulin as loading control. (F) Quantification of experiments as depicted in (E) were performed by comparing the amount of coimmunoprecipitated RAB7L1 to the amount of LRRK2 in the immunoprecipitation (IP). Data are mean ± SEM ( n = 6 independent experiments).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: RAB7L1-Mediated Relocalization of LRRK2 to the Golgi Complex Causes Centrosomal Deficits via RAB8A

    doi: 10.3389/fnmol.2018.00417

    Figure Lengend Snippet: RAB7L1 causes recruitment of LRRK2 in a manner independent of kinase activity. (A) Example of HEK293T cells transfected with either GFP-tagged wildtype LRRK2 (green), or cotransfected with wildtype LRRK2 and wildtype or mutant mRFP-tagged RAB7L1 (red) as indicated, and stained with DAPI (blue). Scale bar, 10 μm. (B) Example of cells cotransfected with the indicated pathogenic GFP-LRRK2 mutants (green) and mRFP-RAB7L1 (red) as indicated, and stained with DAPI (blue). Scale bar, 10 μm. (C) Example of cells cotransfected with mRFP-RAB7L1 (red) and either wildtype or K1906M kinase-dead mutant GFP-LRRK2 (green), or cotransfected with wildtype GFP-LRRK2 (green) and treated with 500 nM LRRK2-IN-1 (INH1) or 500 nM GSK2578215A (GSK) for 60 min before fixation, and stained with DAPI (blue). Scale bar, 10 μm. (D) Quantification of the percentage of colocalization of the indicated LRRK2 constructs with RAB7L1 as determined by analysis of Mander’s coefficient (M2 × 100) from 10 random cells per condition. (E) Cells were cotransfected with the indicated GFP-tagged LRRK2 constructs and flag-tagged RAB7L1, and treated with 100 nM MLi2 for 60 min where indicated. Binding between LRRK2 and RAB7L1 was assessed by co-immunoprecipitation of GFP-tagged LRRK2 variants with flag-tagged RAB7L1 using a polyclonal GFP antibody. Left panel shows inputs, and right panel shows samples after immunoprecipitation, probed either for GFP (using a monoclonal GFP antibody), or in a separate gel probed for flag and tubulin as loading control. (F) Quantification of experiments as depicted in (E) were performed by comparing the amount of coimmunoprecipitated RAB7L1 to the amount of LRRK2 in the immunoprecipitation (IP). Data are mean ± SEM ( n = 6 independent experiments).

    Article Snippet: Protein concentration of supernatants was estimated using the BCA assay (Pierce), and 30 μg of extracts resolved by SDS-PAGE and analyzed by Western blot, using a rabbit polyclonal anti-GFP antibody (ab6556, 1:3000, Abcam), a knockout-validated sheep polyclonal anti-RAB8A (MRC PPU, S969D, 1:200), a sheep polyclonal anti-phospho-RAB8A (MRC PPU, S874D, 1:200), a knockout-validated rabbit monoclonal anti-RAB8A (Abcam, ab188574, 1:1000), a knockout-validated sheep polyclonal anti-RAB7L1 (MRC PPU, S984D, 1:250), a rabbit polyclonal anti-T72-phospho-RAB8A (1:500, generous gift of D. Alessi, University of Dundee, United Kingdom), a mouse monoclonal anti-flag antibody (clone M2, 1:2000, Sigma) and a mouse monoclonal anti-GAPDH (ab9484, 1:2000, Abcam) as loading control.

    Techniques: Activity Assay, Transfection, Mutagenesis, Staining, Construct, Binding Assay, Immunoprecipitation

    Expression of GFP in zebrafish embryos injected with HCA-EGFP. HCA-EGFP was injected in the brain of zebrafish embryos at 48 hpf. ( A ) Representative images of zebrafish embryos photographed 48 h later using a fluorescence stereomicroscope. A control embryo not injected with the vector is included to show autofluorescence (100x). ( B ) Immunohistochemistry against GFP (100× and 200× magnification, as indicated).

    Journal: Genes

    Article Title: Danio Rerio as Model Organism for Adenoviral Vector Evaluation

    doi: 10.3390/genes10121053

    Figure Lengend Snippet: Expression of GFP in zebrafish embryos injected with HCA-EGFP. HCA-EGFP was injected in the brain of zebrafish embryos at 48 hpf. ( A ) Representative images of zebrafish embryos photographed 48 h later using a fluorescence stereomicroscope. A control embryo not injected with the vector is included to show autofluorescence (100x). ( B ) Immunohistochemistry against GFP (100× and 200× magnification, as indicated).

    Article Snippet: Serial paraffin sections (3-µm thick) were cut, and immunohistochemistry was applied using rabbit anti-GFP (ab6556, Abcam, Cambridge, UK) as the primary antibody (1:2000).

    Techniques: Expressing, Injection, High Content Screening, Fluorescence, Plasmid Preparation, Immunohistochemistry

    Expression of green fluorescent protein (GFP) in zebrafish embryos injected with Ad-EGFP. ( A ) Schematic representation of brain regions: telencephalon (T), mesencephalon (M), and rhombencephalon (R), modified from. The blue arrow represents the injection area. Ad-EGFP was injected in the brain of zebrafish embryos at 48 hpf. ( B ) Representative images of zebrafish embryos photographed 48 h later using a fluorescence stereomicroscope (100x). ( C ) Immunohistochemistry against GFP (100× and 200× magnification, as indicated).

    Journal: Genes

    Article Title: Danio Rerio as Model Organism for Adenoviral Vector Evaluation

    doi: 10.3390/genes10121053

    Figure Lengend Snippet: Expression of green fluorescent protein (GFP) in zebrafish embryos injected with Ad-EGFP. ( A ) Schematic representation of brain regions: telencephalon (T), mesencephalon (M), and rhombencephalon (R), modified from. The blue arrow represents the injection area. Ad-EGFP was injected in the brain of zebrafish embryos at 48 hpf. ( B ) Representative images of zebrafish embryos photographed 48 h later using a fluorescence stereomicroscope (100x). ( C ) Immunohistochemistry against GFP (100× and 200× magnification, as indicated).

    Article Snippet: Serial paraffin sections (3-µm thick) were cut, and immunohistochemistry was applied using rabbit anti-GFP (ab6556, Abcam, Cambridge, UK) as the primary antibody (1:2000).

    Techniques: Expressing, Injection, Modification, Fluorescence, Immunohistochemistry

    Regulation of autophagy by MC159 and its SH3BP4 binding-deficient mutant. (A and B) Control MCF-7/LC3-EGFP cells or their lentivirally transduced derivatives stably expressing wild-type MC159 or MC159 AXXA(N) were examined under normal culture conditions (A) or after 6 h of starvation (B) in medium lacking amino acids and serum using fluorescence microscopy imaging of nuclear (Hoechst)-, LC3-, and MC159-specific signals, as indicated. (C) Uniform LC3-GFP expression in all cells, equal MC159 expression in the transduced cells, and the lack of SH3BP4 expression in the knockout cells were verified by Western blotting using antibodies against GFP, mCherry, and SH3BP4, respectively. (D) Decrease of p62 expression in MCF-7/LC3-EGFP cells (control, C) and their derivatives expressing wild-type MC159 (WT) or MC159 AXXA(N) (AXXA) was examined by Western blotting. The anti-p62 signals were quantified and normalized to levels of the corresponding loading control values (anti-α-tubulin signal) to calculate the relative decrease in p62 expression, which is shown as a bar graph above the blot. (E) Box blot presentation of the data from automated quantification of the number of autophagosomes in the parental MCF-7/LC3-EGFP cells and their SH3BP4 knockout derivatives used as controls (C) or transduced with wild-type MC159 (WT) or MC159 AXXA(N) (AXXA), as indicated. The boxes mark the two middle quartiles of the data points separated by a line showing the median autophagosome count, and the whiskers show the distribution of all values in the upper and lower data point quartiles. The statistical significance of the indicated comparisons is shown as follows: *, P = 6,291 × 10 −93 ; **, P = 5,868 × 10 −101 .

    Journal: Journal of Virology

    Article Title: MC159 of Molluscum Contagiosum Virus Suppresses Autophagy by Recruiting Cellular SH3BP4 via an SH3 Domain-Mediated Interaction

    doi: 10.1128/JVI.01613-18

    Figure Lengend Snippet: Regulation of autophagy by MC159 and its SH3BP4 binding-deficient mutant. (A and B) Control MCF-7/LC3-EGFP cells or their lentivirally transduced derivatives stably expressing wild-type MC159 or MC159 AXXA(N) were examined under normal culture conditions (A) or after 6 h of starvation (B) in medium lacking amino acids and serum using fluorescence microscopy imaging of nuclear (Hoechst)-, LC3-, and MC159-specific signals, as indicated. (C) Uniform LC3-GFP expression in all cells, equal MC159 expression in the transduced cells, and the lack of SH3BP4 expression in the knockout cells were verified by Western blotting using antibodies against GFP, mCherry, and SH3BP4, respectively. (D) Decrease of p62 expression in MCF-7/LC3-EGFP cells (control, C) and their derivatives expressing wild-type MC159 (WT) or MC159 AXXA(N) (AXXA) was examined by Western blotting. The anti-p62 signals were quantified and normalized to levels of the corresponding loading control values (anti-α-tubulin signal) to calculate the relative decrease in p62 expression, which is shown as a bar graph above the blot. (E) Box blot presentation of the data from automated quantification of the number of autophagosomes in the parental MCF-7/LC3-EGFP cells and their SH3BP4 knockout derivatives used as controls (C) or transduced with wild-type MC159 (WT) or MC159 AXXA(N) (AXXA), as indicated. The boxes mark the two middle quartiles of the data points separated by a line showing the median autophagosome count, and the whiskers show the distribution of all values in the upper and lower data point quartiles. The statistical significance of the indicated comparisons is shown as follows: *, P = 6,291 × 10 −93 ; **, P = 5,868 × 10 −101 .

    Article Snippet: ; Cell Signaling Technology, MA, USA) rabbit anti-LC3B (D11) XP (Cell Signaling Technology, MA, USA), mouse monoclonal (6G6) to red fluorescent protein (RFP) (ChromoTek), rabbit anti-GFP (sc-8334; Santa Cruz Biotechnology), and rabbit anti-p62 (Enzo).

    Techniques: Binding Assay, Mutagenesis, Stable Transfection, Expressing, Fluorescence, Microscopy, Imaging, Knock-Out, Western Blot, Transduction

    A. Delineation of the smallest portion of Ge-1 that mediates interaction with LMKB. GFP-NLS-Ge-1 fragment (amino acids 630–1437) localized to nuclear dots (green, ii) in HEp-2 cells, but RFP-LMKB remained in P-bodies and was not detected in the nucleus (red, i), suggesting that N-terminal amino acids in Ge-1 are required for interaction with LMKB. To identify the N-terminal portion of Ge-1 that interacts with LMKB, RFP-LMKB was tested for the ability to recruit N-terminal fragments of Ge-1 to P-bodies. In the presence of RFP-LMKB (red, iv), GFP-Ge-1(1–1094) localized to cytoplasmic dots resembling P-bodies (green, v). In cells expressing RFP-LMKB (red, vii) and GFP-Ge-1(1–1094) (green, viii), both proteins co-localized with endogenous Ge-1 (blue, ix), confirming that these structures are P-bodies. In the absence of LMKB (RFP alone, red, x), GFP-Ge-1(1–1094) did not localize to P-bodies (green, xi), but was instead distributed throughout the cytoplasm. Smaller fragments of Ge-1, including amino acids 1–935 (green, xiv) and 104–1094 (green, xvii) did not co-localize with co-expressed LMKB in P-bodies. Merge of fluorescence in i and ii, iv and v, x and xi, xiii and xiv, and xvi and xvii is shown in iii, vi, xii, xv and xviii. DAPI staining (blue in the merged panels) indicates the location of nuclei. White bar in xviii indicates 5.0 µm. B. Schematic representation of Ge-1 and summary of results. The N-terminus of Ge-1 contains a WD40 repeat domain. The C-terminus contains four regions that have repeating hydrophobic residue periodicity (ψ(X 2-3 )-repeat domains). + indicates a fragment of Ge-1 that interacts with LMKB; - indicates a fragment of Ge-1 that does not interact with LMKB. The black rectangle indicates the smallest tested portion of Ge-1 that interacts with LMKB.

    Journal: PLoS ONE

    Article Title: LMKB/MARF1 Localizes to mRNA Processing Bodies, Interacts with Ge-1, and Regulates IFI44L Gene Expression

    doi: 10.1371/journal.pone.0094784

    Figure Lengend Snippet: A. Delineation of the smallest portion of Ge-1 that mediates interaction with LMKB. GFP-NLS-Ge-1 fragment (amino acids 630–1437) localized to nuclear dots (green, ii) in HEp-2 cells, but RFP-LMKB remained in P-bodies and was not detected in the nucleus (red, i), suggesting that N-terminal amino acids in Ge-1 are required for interaction with LMKB. To identify the N-terminal portion of Ge-1 that interacts with LMKB, RFP-LMKB was tested for the ability to recruit N-terminal fragments of Ge-1 to P-bodies. In the presence of RFP-LMKB (red, iv), GFP-Ge-1(1–1094) localized to cytoplasmic dots resembling P-bodies (green, v). In cells expressing RFP-LMKB (red, vii) and GFP-Ge-1(1–1094) (green, viii), both proteins co-localized with endogenous Ge-1 (blue, ix), confirming that these structures are P-bodies. In the absence of LMKB (RFP alone, red, x), GFP-Ge-1(1–1094) did not localize to P-bodies (green, xi), but was instead distributed throughout the cytoplasm. Smaller fragments of Ge-1, including amino acids 1–935 (green, xiv) and 104–1094 (green, xvii) did not co-localize with co-expressed LMKB in P-bodies. Merge of fluorescence in i and ii, iv and v, x and xi, xiii and xiv, and xvi and xvii is shown in iii, vi, xii, xv and xviii. DAPI staining (blue in the merged panels) indicates the location of nuclei. White bar in xviii indicates 5.0 µm. B. Schematic representation of Ge-1 and summary of results. The N-terminus of Ge-1 contains a WD40 repeat domain. The C-terminus contains four regions that have repeating hydrophobic residue periodicity (ψ(X 2-3 )-repeat domains). + indicates a fragment of Ge-1 that interacts with LMKB; - indicates a fragment of Ge-1 that does not interact with LMKB. The black rectangle indicates the smallest tested portion of Ge-1 that interacts with LMKB.

    Article Snippet: GFP, GFP-LMKB(1457–1742) and GFP-LMKB(1622–1742) were detected using rabbit anti-GFP antibodies.

    Techniques: Expressing, Fluorescence, Staining

    Indirect immunofluorescence shows that LMKB localizes to P-bodies. GFP-LMKB (green, i) localized to discrete, dot-like structures in the cytoplasm of transfected HEp-2 cells and co-localized with co-expressed FLAG-Ge-1 (red, ii). To determine the cellular location of endogenous LMKB, rabbit anti-LMKB antiserum was used to stain Hut78 cells. LMKB (green, iv) co-localized with Ge-1 (red, v), identified using human serum 0121. After exposure to arsenite for 1 hour, TIA (a marker of stress granules) was detected in cytoplasmic granules (red, viii). LMKB (green, vii) did not co-localize with TIA in stress granules, but was instead detected in adjacent P-bodies. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining in iii and vi (blue) indicates the location of nuclei. White arrows in vii and ix indicate representative LMKB-containing P-bodies adjacent to stress granules. White bar in ix indicates 5.0 µm.

    Journal: PLoS ONE

    Article Title: LMKB/MARF1 Localizes to mRNA Processing Bodies, Interacts with Ge-1, and Regulates IFI44L Gene Expression

    doi: 10.1371/journal.pone.0094784

    Figure Lengend Snippet: Indirect immunofluorescence shows that LMKB localizes to P-bodies. GFP-LMKB (green, i) localized to discrete, dot-like structures in the cytoplasm of transfected HEp-2 cells and co-localized with co-expressed FLAG-Ge-1 (red, ii). To determine the cellular location of endogenous LMKB, rabbit anti-LMKB antiserum was used to stain Hut78 cells. LMKB (green, iv) co-localized with Ge-1 (red, v), identified using human serum 0121. After exposure to arsenite for 1 hour, TIA (a marker of stress granules) was detected in cytoplasmic granules (red, viii). LMKB (green, vii) did not co-localize with TIA in stress granules, but was instead detected in adjacent P-bodies. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining in iii and vi (blue) indicates the location of nuclei. White arrows in vii and ix indicate representative LMKB-containing P-bodies adjacent to stress granules. White bar in ix indicates 5.0 µm.

    Article Snippet: GFP, GFP-LMKB(1457–1742) and GFP-LMKB(1622–1742) were detected using rabbit anti-GFP antibodies.

    Techniques: Immunofluorescence, Transfection, Staining, Marker, Fluorescence

    A. A modified two-hybrid assay was used to test for interaction between LMKB and Ge-1 and to identify the portion of LMKB that mediates interaction with Ge-1. Expression of a plasmid encoding Ge-1 fused to a nuclear localization sequence (NLS) shifted some of the protein from the cytoplasm to the nucleus of HEp-2 cells (red, i, iv and vii), where it localized to dot-like structures. Co-transfection of plasmids encoding GFP fused to full-length LMKB (green, ii) and NLS-Ge-1 resulted in co-localization of the two proteins in nuclear dots. GFP-LMKB fragment fusion proteins encoding amino acids 1622–1742 (green, v) also co-localized with co-expressed NLS-Ge-1 (red, iv) in nuclear dots. GFP-LMKB fragments encoding 1–687 and 389–1199 (both not shown) and 1–1622 (green, viii) did not co-localize with NLS-Ge-1 in nuclear dots. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining (blue) in iii, vi and ix indicates the location of nuclei. Human serum 0121 containing anti-Ge-1 antibodies and mouse monoclonal anti-GFP antibodies were used to detect Ge-1 and GFP, respectively. White arrows in vii and ix indicate nuclear dots containing NLS-Ge-1. White bar in ix indicates 5.0 µm. B . Schematic representation of the structure of LMKB and summary of results. LMKB contains an N-terminal globular domain (“LK”, amino acids 351–493) that may serve as a cation binding domain. Two RRM-type RNA binding domains are located between amino acids 510–600 and 792–867. The C-terminus of LMKB contains eight helix-turn-helix folds (“OST domains”) that are predicted to bind to dsRNA (amino acids 879–937, 1004–1074, 1100–1170, 1176–1247, 1259–1330, 1337–1406, 1412–1484, 1487–1557; numbering system is as indicated in GenBank #NM_014647). + indicates an interaction between a LMKB fragment and Ge-1; - indicates a fragment of LMKB that does not interact with Ge-1. The black rectangle indicates the smallest tested portion of LMKB that interacts with Ge-1.

    Journal: PLoS ONE

    Article Title: LMKB/MARF1 Localizes to mRNA Processing Bodies, Interacts with Ge-1, and Regulates IFI44L Gene Expression

    doi: 10.1371/journal.pone.0094784

    Figure Lengend Snippet: A. A modified two-hybrid assay was used to test for interaction between LMKB and Ge-1 and to identify the portion of LMKB that mediates interaction with Ge-1. Expression of a plasmid encoding Ge-1 fused to a nuclear localization sequence (NLS) shifted some of the protein from the cytoplasm to the nucleus of HEp-2 cells (red, i, iv and vii), where it localized to dot-like structures. Co-transfection of plasmids encoding GFP fused to full-length LMKB (green, ii) and NLS-Ge-1 resulted in co-localization of the two proteins in nuclear dots. GFP-LMKB fragment fusion proteins encoding amino acids 1622–1742 (green, v) also co-localized with co-expressed NLS-Ge-1 (red, iv) in nuclear dots. GFP-LMKB fragments encoding 1–687 and 389–1199 (both not shown) and 1–1622 (green, viii) did not co-localize with NLS-Ge-1 in nuclear dots. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining (blue) in iii, vi and ix indicates the location of nuclei. Human serum 0121 containing anti-Ge-1 antibodies and mouse monoclonal anti-GFP antibodies were used to detect Ge-1 and GFP, respectively. White arrows in vii and ix indicate nuclear dots containing NLS-Ge-1. White bar in ix indicates 5.0 µm. B . Schematic representation of the structure of LMKB and summary of results. LMKB contains an N-terminal globular domain (“LK”, amino acids 351–493) that may serve as a cation binding domain. Two RRM-type RNA binding domains are located between amino acids 510–600 and 792–867. The C-terminus of LMKB contains eight helix-turn-helix folds (“OST domains”) that are predicted to bind to dsRNA (amino acids 879–937, 1004–1074, 1100–1170, 1176–1247, 1259–1330, 1337–1406, 1412–1484, 1487–1557; numbering system is as indicated in GenBank #NM_014647). + indicates an interaction between a LMKB fragment and Ge-1; - indicates a fragment of LMKB that does not interact with Ge-1. The black rectangle indicates the smallest tested portion of LMKB that interacts with Ge-1.

    Article Snippet: GFP, GFP-LMKB(1457–1742) and GFP-LMKB(1622–1742) were detected using rabbit anti-GFP antibodies.

    Techniques: Modification, Two Hybrid Assay, Expressing, Plasmid Preparation, Sequencing, Cotransfection, Fluorescence, Staining, Binding Assay, RNA Binding Assay

    Ge-1 co-precipitates with C-terminal fragments of LMKB. COS-7 cells were transfected with plasmids encoding FLAG-Ge-1, GFP-LMKBc (containing amino acids 1457–1742), GFP-LMKBc’ (amino acids 1622–1742), FLAG and GFP as indicated. Extracts were prepared and incubated with mouse anti-GFP antibody and protein G coupled to Sepharose beads. Precipitates (top two panels) are compared with 5% of the total COS-7 cell extract input (bottom two panels). FLAG-Ge-1 co-precipitated with GFP-LMKBc(1457–1742) and with GFP-LMKBc’(1622–1742), but not with GFP alone.

    Journal: PLoS ONE

    Article Title: LMKB/MARF1 Localizes to mRNA Processing Bodies, Interacts with Ge-1, and Regulates IFI44L Gene Expression

    doi: 10.1371/journal.pone.0094784

    Figure Lengend Snippet: Ge-1 co-precipitates with C-terminal fragments of LMKB. COS-7 cells were transfected with plasmids encoding FLAG-Ge-1, GFP-LMKBc (containing amino acids 1457–1742), GFP-LMKBc’ (amino acids 1622–1742), FLAG and GFP as indicated. Extracts were prepared and incubated with mouse anti-GFP antibody and protein G coupled to Sepharose beads. Precipitates (top two panels) are compared with 5% of the total COS-7 cell extract input (bottom two panels). FLAG-Ge-1 co-precipitated with GFP-LMKBc(1457–1742) and with GFP-LMKBc’(1622–1742), but not with GFP alone.

    Article Snippet: GFP, GFP-LMKB(1457–1742) and GFP-LMKB(1622–1742) were detected using rabbit anti-GFP antibodies.

    Techniques: Transfection, Incubation

    H2A.Z acidic patch is incorporated at lower levels at target genes. ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z WT , H2A.Z AP3 , H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z WT , H2A.Z AP3 , and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z WT , H2A.Z AP3 , and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z WT and H2A.Z AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z WT and H2A.Z AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z WT in chromatin fractions compared to H2A.Z AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z WT and H2A.Z AP3 . Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z AP3 . (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z WT and H2A.Z AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z WT and H2A.Z AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.

    Journal: PLoS Genetics

    Article Title: H2A.Z Acidic Patch Couples Chromatin Dynamics to Regulation of Gene Expression Programs during ESC Differentiation

    doi: 10.1371/journal.pgen.1003725

    Figure Lengend Snippet: H2A.Z acidic patch is incorporated at lower levels at target genes. ChIP-Seq analysis of H2A.Z in ESCs shows that the divergent acidic patch residues are required for stable incorporation of H2A.Z (A) Density map of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) enrichment at all H2A.Z target genes ordered from most H3K27me3 enriched genes to least H3K27me3 enriched genes in ESCs within the region −4 kb to +4 kb relative to the TSS. The right panel representing the expression levels of the corresponding genes in ESCs generated from RNA-Seq data. Red to white gradient represents genes with high to low expression levels respectively. (B) Average enrichment patterns of H2A.Z WT , H2A.Z AP3 , H3K4me3, H3K27me3, and RNAP2-Ser5P +/−2 kb around the TSS at bivalent (top) and H3K4me3 (H3K27me3 negative) only promoters (bottom). H2A.Z WT , H2A.Z AP3 , and H3K27me3 are plotted on the primary axis (right). H3K4me3 and RNAP2-Ser5P are plotted on the secondary axis (left). (C) Average read density plots comparing binding profiles of H2A.Z WT , H2A.Z AP3 , and input at all H2A.Z target gene promoters in ESCs plotted +/−2 kb relative to TSS. The ChIP-Seq datasets for H2A.Z WT and H2A.Z AP3 were generated using GFP antibodies against the YFP transgene. (D) Genome profile of ChIP-Seq reads showing the distribution of H2A.Z WT (dark blue), H2A.Z AP3 (light blue), H3K4me3 (red), and H3K27me3 (light green) across the HoxA locus- a representative set of H2A.Z target genes. (E) Semi-quantitative western blot of H2A.Z WT and H2A.Z AP3 chromatin fractions probed with GFP and H3 (load control) using a range of DNA concentrations (top). Graph quantifying the ratio of transgene levels relative to H3 at the indicated DNA concentrations shows ∼1.85 fold more H2A.Z WT in chromatin fractions compared to H2A.Z AP3 (bottom). Fold change was calculated from the average ratio of each transgene to H3. Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.255) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) for replicate 1 (R1) were used to calculate the 1.72 (0.439/0.255) fold change between H2A.Z WT and H2A.Z AP3 . Similar results were obtained for an independent replicate (R2). Ratios for H2A.Z WT /H3 (0.439) and H2A.Z AP3 /H3 (0.219) at the two intermediate DNA concentrations (160 µg/µl and 240 µg/µl) were used to calculate the 2.0 (0.439/0.219) for R2. Thus, the levels of H2A.Z WT were on average 1.85-fold higher in chromatin-associated fractions relative to H2A.Z AP3 . (F) Graph showing the ratio of SRCAP and RUVBL1 signal to their respective input signal, from co-immunoprecipitation analyses performed in H2A.Z WT and H2A.Z AP3 ESCs (in the endogenous H2A.Z knockdown background). Densitometric measurements of the western blots were performed in ImageJ. The standard deviations were generated from triplicates data points. (G) Nuclei isolated from H2A.Z WT and H2A.Z AP3 expressing ESCs were subjected to increasing salt concentrations as indicated. Histones were extracted at these salt concentrations and resolved by SDS-PAGE. Histones were detected by immunoblotting with GFP antibodies.

    Article Snippet: Rabbit anti-H2A.Z antibody (Abcam, ab4174) and Rabbit anti-GFP antibody (Abcam, ab290) was used for western blot at concentrations recommended by the manufacturer.

    Techniques: Chromatin Immunoprecipitation, Expressing, Generated, RNA Sequencing Assay, Binding Assay, Western Blot, Immunoprecipitation, Isolation, SDS Page

    Cellular assays of p62 polymeric state. a Schematic illustration of used p62 constructs and chimeras with p62-PB1 (green) and TFG1–PB1 (blue). b Representative, negatively stained electron micrographs of purified p62 constructs and chimeras from ( a ), including illustration of polymeric and oligomeric forms observed by negative staining electron microscopy. c Confocal fluorescent images of HeLa p62 (KO) cells expressing GFP-tagged constructs and chimeras. All examined constructs form punctate structures. d Quantification of the number of p62 bodies forming dots of various sizes. e Quantification of cells displaying yellow and red dots in ( f ). f Representative confocal fluorescence images of HeLa p62 (KO) cells expressing mCherry-YFP-tagged (dt-tagged) p62 constructs and chimeras. The appearance of red puncta (as an indicator of lysosomal localization) for all constructs indicates that all constructs and chimeras can be processed by autophagy. Punctae were counted and classified based on more than 100 cells in each condition in three independent experiments. g Representative confocal fluorescence images of HeLa p62 (KO) cells expressing the respective p62 constructs and chimeras, as well as mCherry-YFP-tagged KEAP1. h Statistics of appearance of lysosome-localized and cytosolic dots for mCherry-YFP-tagged KEAP1. The error bars in d , e , and h represent standard deviations of the mean.

    Journal: Nature Communications

    Article Title: Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake

    doi: 10.1038/s41467-020-14343-8

    Figure Lengend Snippet: Cellular assays of p62 polymeric state. a Schematic illustration of used p62 constructs and chimeras with p62-PB1 (green) and TFG1–PB1 (blue). b Representative, negatively stained electron micrographs of purified p62 constructs and chimeras from ( a ), including illustration of polymeric and oligomeric forms observed by negative staining electron microscopy. c Confocal fluorescent images of HeLa p62 (KO) cells expressing GFP-tagged constructs and chimeras. All examined constructs form punctate structures. d Quantification of the number of p62 bodies forming dots of various sizes. e Quantification of cells displaying yellow and red dots in ( f ). f Representative confocal fluorescence images of HeLa p62 (KO) cells expressing mCherry-YFP-tagged (dt-tagged) p62 constructs and chimeras. The appearance of red puncta (as an indicator of lysosomal localization) for all constructs indicates that all constructs and chimeras can be processed by autophagy. Punctae were counted and classified based on more than 100 cells in each condition in three independent experiments. g Representative confocal fluorescence images of HeLa p62 (KO) cells expressing the respective p62 constructs and chimeras, as well as mCherry-YFP-tagged KEAP1. h Statistics of appearance of lysosome-localized and cytosolic dots for mCherry-YFP-tagged KEAP1. The error bars in d , e , and h represent standard deviations of the mean.

    Article Snippet: Autophagy and p62 turnover assays The following antibodies were used: mouse anti-Myc antibody (Cell Signaling, Cat. #2276#, 1:8000 for western blots and 1:5000 for confocal imaging); rabbit anti-GFP antibody (Abcam, ab290, 1:5000); guinea pig anti-p62 antibody (Progen, Cat. #Gp62-C#, 1:5000); rabbit anti-actin antibody (Sigma, Cat. #A2066#, 1:1000); Alexa Fluor® 647-conjugated goat anti-mouse IgG ( A21236 , 1:1000); HRP-conjugated goat anti-mouse IgG (1:5000); goat anti-rabbit IgG (1:5000); goat anti-guinea pig IgG (1:5000).

    Techniques: Construct, Staining, Purification, Negative Staining, Electron Microscopy, Expressing, Fluorescence

    Molecular weight patterns of over-expressed MOS1 fusion in HeLa cells. Proteins extracted from HeLa cells over-expressing V5-MOS1 or MOS1V2-GFP were analyzed by immunoblotting after separation by polyacrylamide gel electrophoresis. V5-MOS1 and MOS1V2-GFP were repectively revealed by first hybridizing a mouse anti-V5 monoclonal antibody or a rabbit polyclonal anti-GFP a mouse a rabbit polyclonal anti-GFP. The filters were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG, followed by development using enhanced chemiluminescence.

    Journal: PLoS ONE

    Article Title: Nuclear Importation of Mariner Transposases among Eukaryotes: Motif Requirements and Homo-Protein Interactions

    doi: 10.1371/journal.pone.0023693

    Figure Lengend Snippet: Molecular weight patterns of over-expressed MOS1 fusion in HeLa cells. Proteins extracted from HeLa cells over-expressing V5-MOS1 or MOS1V2-GFP were analyzed by immunoblotting after separation by polyacrylamide gel electrophoresis. V5-MOS1 and MOS1V2-GFP were repectively revealed by first hybridizing a mouse anti-V5 monoclonal antibody or a rabbit polyclonal anti-GFP a mouse a rabbit polyclonal anti-GFP. The filters were then incubated with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG, followed by development using enhanced chemiluminescence.

    Article Snippet: After blocking with 5% skim milk in phosphate-buffered saline for 1 h, the filters were incubated overnight with a mouse anti-V5 monoclonal antibody or a rabbit polyclonal anti-GFP (1∶2000 for both; invitrogen).

    Techniques: Molecular Weight, Expressing, Polyacrylamide Gel Electrophoresis, Incubation

    Silencing calcineurin expression and a pharmacological inhibitor of calcineurin decrease 5-HT uptake via SERT. A , HEK-293 cells were transfected with the plasmid encoding SERT and control or CaNA siRNA. Total protein extracts were analyzed by Western blotting using the monoclonal anti-GFP antibody, the polyclonal anti-CaNA antibody, and the anti-GAPDH antibody. B , [ 3 H]-5-HT uptake in HEK-293 cells expressing SERT in the presence of control or CaNA siRNA. A representative experiment is shown. Data for V max of 5-HT uptake, expressed in percentage of value in cells transfected with control siRNA (3.78 ± 0.49 pmol/min), are the mean ± SEM of values measured in three independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: Calcineurin Interacts with the Serotonin Transporter C-Terminus to Modulate Its Plasma Membrane Expression and Serotonin Uptake

    doi: 10.1523/JNEUROSCI.0076-13.2013

    Figure Lengend Snippet: Silencing calcineurin expression and a pharmacological inhibitor of calcineurin decrease 5-HT uptake via SERT. A , HEK-293 cells were transfected with the plasmid encoding SERT and control or CaNA siRNA. Total protein extracts were analyzed by Western blotting using the monoclonal anti-GFP antibody, the polyclonal anti-CaNA antibody, and the anti-GAPDH antibody. B , [ 3 H]-5-HT uptake in HEK-293 cells expressing SERT in the presence of control or CaNA siRNA. A representative experiment is shown. Data for V max of 5-HT uptake, expressed in percentage of value in cells transfected with control siRNA (3.78 ± 0.49 pmol/min), are the mean ± SEM of values measured in three independent experiments. ** p

    Article Snippet: The mouse monoclonal anti-GFP antibody (mixture of clones 7.1 and 13.1) was from Roche Diagnostics, the rabbit polyclonal anti-GFP antibody from Invitrogen, the rabbit polyclonal anti-SERT antibody from ImmunoStar, the rabbit polyclonal anti-CaNA and CaNB antibody from Millipore Bioscience Research Reagents, the rabbit polyclonal anti-CaNA antibody from Millipore, the mouse monoclonal anti-CaNA antibody and the polyclonal anti-Flag antibody from Sigma-Aldrich, and the rabbit anti-GAPDH antibody from Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot

    Increased TCTP secretion by over-expression of proton pump is inhibited by pantoprazole in HEK293. (A) Comparison of protein expressions in control and Hαβ samples. Control sample was transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct. Hαβ sample was transfected with rat H + /K + -ATPase α1-GFP, HA-rat Na + /K + -ATPase β1, and TCTP-3Xflag constructs. WB: anti-GFP Ab (purified rabbit polyclonal antibody, InVitrogen), anti-HA Ab (mouse 12CA5 monoclonal antibody, Santa Cruz), and anti-flag Ab, (B) TCTP secretion is increased by over-expression of H + /K + -ATPase α1 and Na + /K + -ATPase β1. Both groups of cells were incubated for 3 h in conditioned media. WB: anti-flag Ab, (C) The secretion assay data for the cells transfected with H + /K + -ATPase α1, Na + /K + -ATPase β1, and TCTP. Pantoprazole (1 mM) or omeprazole (1 mM) was treated during 3 h secretion assay. ‘O’ in the graph means omeprazole and ‘P’ does pantoprazole. WB: anti-flag Ab. Data of panel B and panel C represent means±S.D. from three independent experiments. #: inhibition, p

    Journal: PLoS ONE

    Article Title: Proton Pump Inhibitors Exert Anti-Allergic Effects by Reducing TCTP Secretion

    doi: 10.1371/journal.pone.0005732

    Figure Lengend Snippet: Increased TCTP secretion by over-expression of proton pump is inhibited by pantoprazole in HEK293. (A) Comparison of protein expressions in control and Hαβ samples. Control sample was transfected with two empty vectors (pEGFP-N1 and pcDNAI-neo) and TCTP-3Xflag construct. Hαβ sample was transfected with rat H + /K + -ATPase α1-GFP, HA-rat Na + /K + -ATPase β1, and TCTP-3Xflag constructs. WB: anti-GFP Ab (purified rabbit polyclonal antibody, InVitrogen), anti-HA Ab (mouse 12CA5 monoclonal antibody, Santa Cruz), and anti-flag Ab, (B) TCTP secretion is increased by over-expression of H + /K + -ATPase α1 and Na + /K + -ATPase β1. Both groups of cells were incubated for 3 h in conditioned media. WB: anti-flag Ab, (C) The secretion assay data for the cells transfected with H + /K + -ATPase α1, Na + /K + -ATPase β1, and TCTP. Pantoprazole (1 mM) or omeprazole (1 mM) was treated during 3 h secretion assay. ‘O’ in the graph means omeprazole and ‘P’ does pantoprazole. WB: anti-flag Ab. Data of panel B and panel C represent means±S.D. from three independent experiments. #: inhibition, p

    Article Snippet: Antibodies Mouse 12CA5 anti-HA monoclonal antibody, purified rabbit anti-GFP polyclonal antibody, mouse anti-Na+ /K+ -ATPase α1 monoclonal antibody (C464.6), and anti-flag® M2 monoclonal antibody were purchased from Zymed Laboratories Inc., InVitrogen, Upstate, and Sigma, respectively.

    Techniques: Over Expression, Transfection, Construct, Western Blot, Purification, Incubation, Inhibition

    ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p

    Journal: Cell Host & Microbe

    Article Title: A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability

    doi: 10.1016/j.chom.2019.05.007

    Figure Lengend Snippet: ALI Transwells support genetic crosses of C. parvum in vitro (A) Diagram of targeting construct designed to replace the endogenous tk locus (cgd5_4440) with GFP and Nluc-P2A-Neo R cassette. (B) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 4 days post infection (dpi). Transwells were infected with ∼1 × 10 7 unfiltered sporozoites that were electroporated with the TK-GFP-Nluc-P2A-neo-TK reporter and a Cas9 plasmid with a TK gRNA. Transwells were cultured in medium containing PBS (light green) as a control or 20 mM paromomycin (PRM, dark green). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. Nonsignificant (p = 0.11), unpaired Student’s t test between PBS and PRM-treated transwells 4 dpi. (C) Image of whole-mount ALI transwells 5 dpi with transfected C. parvum from same experiment as (B) stained with anti-GFP followed by goat anti-rabbit IgG Alexa Fluor 488. Scale bar, 10 μm. (D) Merged image of (C) with a Pan Cp polyclonal antibody, which recognizes all C. parvum stages, followed by goat antirat IgG Alexa Fluor 568. Scale bar, 10 μm. (E) Diagram of targeting construct designed to replace the endogenous uprt locus (cgd1_1900) with mCherry and Nluc-P2A-Neo R cassette as (A). (F) Relative luminescence normalized to total number of parasites in ALI transwells at 1 and 3 dpi. Transwells were infected with ∼1 × 10 7 unfiltered sporozoites per transwell that were electroporated with the UPRT-mCh-Nluc-P2A-neo-UPRT reporter and a Cas9 plasmid with a UPRT gRNA. Transwells were cultured in medium containing PBS (pink) as a control or 20 mM PRM (red). Data plotted as mean ± S.D. from two transwells per time point from a representative experiment. ∗ p

    Article Snippet: At 1, 3 and 5 dpi, transwells were fixed in 4% formaldehyde and stained for immunohistochemistry as described above using polyclonal rabbit anti-GFP (Thermo Fisher Scientific) with goat anti-rabbit IgG Alexa Fluor 488 secondary and a monoclonal rat anti-mCherry 16D7 (Thermo Fisher Scientific) with goat anti-rat IgG Alexa Fluor 568.

    Techniques: In Vitro, Construct, Infection, Plasmid Preparation, Cell Culture, Transfection, Staining

    Additional verification that charge mutations in the finger loop-proximal region of β-arrestin finger loop produce a constitutive activation phenotype ( a ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and the polar core mutant of β-arrestin-2–GFP (green) in the absence of agonist treatment. ( b ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonist treatment. Statistical significance was calculated using an two-tailed unpaired t test with Welch’s correction (polar core mutant: n=12 cells from 3 independent experiments, p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Additional verification that charge mutations in the finger loop-proximal region of β-arrestin finger loop produce a constitutive activation phenotype ( a ) Live cell TIRF microscopy images showing FLAG–β2AR, clathrin-light-chain–DsRed (red), and the polar core mutant of β-arrestin-2–GFP (green) in the absence of agonist treatment. ( b ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonist treatment. Statistical significance was calculated using an two-tailed unpaired t test with Welch’s correction (polar core mutant: n=12 cells from 3 independent experiments, p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Microscopy, Mutagenesis, Construct, Two Tailed Test

    β-arrestin activation is inhibited by a polar network in a region proximal to the β-arrestin finger loop ( a ) Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR (blue), clathrin-light-chain–DsRed (red), and wild-type (top, green) or finger loop proximal mutant (bottom, green) β-arrestin-2–GFP without agonist treatment. Clustering index measuring constitutive activation of the indicated ( b ) β-arrestin-2–GFP or ( c ) β-arrestin-1–mVenus constructs without agonist treatment (n=12 cells from 3 independent experiments, p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: β-arrestin activation is inhibited by a polar network in a region proximal to the β-arrestin finger loop ( a ) Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR (blue), clathrin-light-chain–DsRed (red), and wild-type (top, green) or finger loop proximal mutant (bottom, green) β-arrestin-2–GFP without agonist treatment. Clustering index measuring constitutive activation of the indicated ( b ) β-arrestin-2–GFP or ( c ) β-arrestin-1–mVenus constructs without agonist treatment (n=12 cells from 3 independent experiments, p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Microscopy, Mutagenesis, Construct

    Direct interaction with the GPCR, but not the GPCR cytoplasmic tail, is required for β-arrestin trafficking activation ( a ) Live cell TIRF microscopy images showing FLAG–β1AR truncated at the 415 th amino acid (415T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs after 10 μM isoproterenol for cells co-expressing the indicated FLAG–β1AR receptor (n=10, 12 cells, respectively, from 3 independent experiments, p=0.5825 calculated using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–β2AR truncated at the 365 th amino acid (365T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells treated with 10 μM isoproterenol and either transfected with FLAG-β2AR or empty vector (n=11, 13 cells, respectively, from 3 independent experiments, p=0.0269 calculated using a two-tailed unpaired t test with Welch’s correction). ( e ) Maximum β-arrestin-2–GFP enrichment at CCSs for cells co-expressing the indicated FLAG–β2AR receptor and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments, p=0.0606 calculated using a two-tailed unpaired t test). ( f ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( g ) Initial enrichment in CCSs before 10 μM quinpirole treatment and ( h ) maximum enrichment after 10 μM quinpirole treatment (n=12 cells from 3 independent experiments; p=0.19 and 0.4873, respectively, using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β1AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 5 μM forskolin (fsk) treatment. ( j ) Initial enrichment in CCSs before 5 μM forskolin (fsk) treatment and ( k ) maximum enrichment after 5 μM fsk treatment (n=12 cells from 3 independent experiments; p=0.6325 and 0.0971, respectively, using a two-tailed unpaired t test). ( l ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2–GFP KNC mutant (green) and clathrin-light-chain–DsRed (red before and after 10 μM isoproterenol treatment. ( m ) Initial enrichment in CCSs before 10 μM isoproterenol treatment and ( n ) maximum enrichment after 10 μM isoproterenol (n=9 (WT) or 8 (KNC) cells from 3 independent experiments; p=0.6681( m ) and p=0.001 ( n ) using a two-tailed unpaired t test with Welch’s correction). ( a, c, f, I, l ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Direct interaction with the GPCR, but not the GPCR cytoplasmic tail, is required for β-arrestin trafficking activation ( a ) Live cell TIRF microscopy images showing FLAG–β1AR truncated at the 415 th amino acid (415T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs after 10 μM isoproterenol for cells co-expressing the indicated FLAG–β1AR receptor (n=10, 12 cells, respectively, from 3 independent experiments, p=0.5825 calculated using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–β2AR truncated at the 365 th amino acid (365T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells treated with 10 μM isoproterenol and either transfected with FLAG-β2AR or empty vector (n=11, 13 cells, respectively, from 3 independent experiments, p=0.0269 calculated using a two-tailed unpaired t test with Welch’s correction). ( e ) Maximum β-arrestin-2–GFP enrichment at CCSs for cells co-expressing the indicated FLAG–β2AR receptor and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments, p=0.0606 calculated using a two-tailed unpaired t test). ( f ) Live cell TIRF microscopy images showing FLAG–DRD2 (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( g ) Initial enrichment in CCSs before 10 μM quinpirole treatment and ( h ) maximum enrichment after 10 μM quinpirole treatment (n=12 cells from 3 independent experiments; p=0.19 and 0.4873, respectively, using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β1AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 5 μM forskolin (fsk) treatment. ( j ) Initial enrichment in CCSs before 5 μM forskolin (fsk) treatment and ( k ) maximum enrichment after 5 μM fsk treatment (n=12 cells from 3 independent experiments; p=0.6325 and 0.0971, respectively, using a two-tailed unpaired t test). ( l ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2–GFP KNC mutant (green) and clathrin-light-chain–DsRed (red before and after 10 μM isoproterenol treatment. ( m ) Initial enrichment in CCSs before 10 μM isoproterenol treatment and ( n ) maximum enrichment after 10 μM isoproterenol (n=9 (WT) or 8 (KNC) cells from 3 independent experiments; p=0.6681( m ) and p=0.001 ( n ) using a two-tailed unpaired t test with Welch’s correction). ( a, c, f, I, l ) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Microscopy, Expressing, Two Tailed Test, Transfection, Plasmid Preparation, Mutagenesis

    Verification that the conserved phosphoinositide binding determinant in the β-arrestin C-domain is specifically required for the catalytic trafficking mechanism and operates upstream of clathrin and AP-2 binding interactions Graphical representation of β-arrestin interaction domains without ( a ) and with ( b ) βAR activation by isoproterenol. ( c ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP lipid mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( e ) Representative western blot (from 4 independent experiments) of purified wild-type and lipid mutant versions of β-arrestin-1(1-393) immunoprecipitation with PIP2-coated agarose beads and quantified in ( f ) as percent of input protein (n=4 independent experiments, p=0.0142 using a two-tailed unpaired t test). For gel source data, see Supplementary Figure 1 . ( g ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (F191G, L192G) lipid anchor mutant mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( h ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing the indicated β-arrestin-2–GFP construct and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments; p=0.9227 calculated using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. ( j ) Representative images of HEK 293 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP lipid and CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. Representative β-arrestin images false colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (data shown as mean ± s.e.m.), respectively, from cells co-expressing FLAG–β1ARs (n=12 cells per condition) without isoproterenol treatment ( k–m ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( n–p ), lipid mutant ( q–s ), CCS mutant ( t–v ), and CCS and lipid mutant ( w–y ). Wild-type β-arrestin-2–GFP maximum enrichment at CCSs shown in panels r, u, x is replotted from panel o. Live cell TIRF microscopy images showing cells before and after 10 μM isoproterenol treatment and co-expressing FLAG–β1AR (blue), clathrin-light-chain–DsRed (red), and the following GFP labeled versions of β-arrestin-2 (green): ( z ) wild-type, ( aa ) lipid mutant, and ( ab ) CCS mutant, and ( ac ) CCS and lipid mutant. ( ad ) Live cell TIRF microscopy images showing FLAG-β2AR and the indicated β-arrestin-2-GFP construct in the absence of agonist treatment. ( ae ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonists treatment. Detailed description of β-arrestin mutations are provided in Extended Data Table 1 . ( c, d, g, i, j, k, n, q, t, w, z, aa, ab, ac, ad ) show representative images from 3 independent experiments. For ( r, u, x) n=12 cells from 3 independent experiments; statistical significance was calculated using an unpaired t test with Welch’s correction, p=0.0007, 0.0018, and 0.0012, respectively. For ( ae ), statistical significance was calculated using an unpaired t test with Welch’s correction, n=12 (WT) and 16 (finger loop proximal mutant) from 3 independent experiments, p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Verification that the conserved phosphoinositide binding determinant in the β-arrestin C-domain is specifically required for the catalytic trafficking mechanism and operates upstream of clathrin and AP-2 binding interactions Graphical representation of β-arrestin interaction domains without ( a ) and with ( b ) βAR activation by isoproterenol. ( c ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( d ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP lipid mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( e ) Representative western blot (from 4 independent experiments) of purified wild-type and lipid mutant versions of β-arrestin-1(1-393) immunoprecipitation with PIP2-coated agarose beads and quantified in ( f ) as percent of input protein (n=4 independent experiments, p=0.0142 using a two-tailed unpaired t test). For gel source data, see Supplementary Figure 1 . ( g ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP (F191G, L192G) lipid anchor mutant mutant (green), and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( h ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing the indicated β-arrestin-2–GFP construct and treated with 10 μM isoproterenol (n=12 cells from 3 independent experiments; p=0.9227 calculated using a two-tailed unpaired t test). ( i ) Live cell TIRF microscopy images showing FLAG–β2AR (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. ( j ) Representative images of HEK 293 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP lipid and CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol. Representative β-arrestin images false colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (data shown as mean ± s.e.m.), respectively, from cells co-expressing FLAG–β1ARs (n=12 cells per condition) without isoproterenol treatment ( k–m ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( n–p ), lipid mutant ( q–s ), CCS mutant ( t–v ), and CCS and lipid mutant ( w–y ). Wild-type β-arrestin-2–GFP maximum enrichment at CCSs shown in panels r, u, x is replotted from panel o. Live cell TIRF microscopy images showing cells before and after 10 μM isoproterenol treatment and co-expressing FLAG–β1AR (blue), clathrin-light-chain–DsRed (red), and the following GFP labeled versions of β-arrestin-2 (green): ( z ) wild-type, ( aa ) lipid mutant, and ( ab ) CCS mutant, and ( ac ) CCS and lipid mutant. ( ad ) Live cell TIRF microscopy images showing FLAG-β2AR and the indicated β-arrestin-2-GFP construct in the absence of agonist treatment. ( ae ) Clustering index of β-arrestin-2–GFP for the indicated construct in the absence of agonists treatment. Detailed description of β-arrestin mutations are provided in Extended Data Table 1 . ( c, d, g, i, j, k, n, q, t, w, z, aa, ab, ac, ad ) show representative images from 3 independent experiments. For ( r, u, x) n=12 cells from 3 independent experiments; statistical significance was calculated using an unpaired t test with Welch’s correction, p=0.0007, 0.0018, and 0.0012, respectively. For ( ae ), statistical significance was calculated using an unpaired t test with Welch’s correction, n=12 (WT) and 16 (finger loop proximal mutant) from 3 independent experiments, p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Binding Assay, Activation Assay, Microscopy, Mutagenesis, Western Blot, Purification, Immunoprecipitation, Two Tailed Test, Expressing, Construct, Fluorescence, Labeling

    Additional demonstration that multiple GPCRs can activate the discrete β-arrestin trafficking mechanism ( a ) Live cell TIRF microscopy images showing FLAG–mu opioid receptor (MOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM DAMGO treatment. ( b ) Average FLAG-MOR and β-arrestin-2–GFP enrichment at CCSs after treatment with 10 μM DAMGO (n=12 cells). ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs for HEK 293 cells expressing FLAG-MOR or empty vector and treated with 10 μM DAMGO (n=12 cells per condition from 3 independent experiments; p=

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Additional demonstration that multiple GPCRs can activate the discrete β-arrestin trafficking mechanism ( a ) Live cell TIRF microscopy images showing FLAG–mu opioid receptor (MOR, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM DAMGO treatment. ( b ) Average FLAG-MOR and β-arrestin-2–GFP enrichment at CCSs after treatment with 10 μM DAMGO (n=12 cells). ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs for HEK 293 cells expressing FLAG-MOR or empty vector and treated with 10 μM DAMGO (n=12 cells per condition from 3 independent experiments; p=

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Microscopy, Expressing, Plasmid Preparation

    Phosphoinositide binding is essential for catalytic activation of β-arrestin trafficking but is dispensable for trafficking mediated by the scaffold mechanism ( a ) Live cell microscopy images of HEK 293 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol treatment. ( b ) Normalized plasma membrane (PM) fluorescence of β-arrestin-2–GFP lipid mutant in cells co-expressing FLAG–β2AR-V2R (n=12 cells from 3 independent experiments) when treated with 10 μM isoproterenol. ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing indicated β-arrestin-2–GFP construct before and after activation of FLAG-β2AR-V2R C tail with 10 μM isoproterenol (n=10, 12 cells, respectively, from 3 independent experiments; p=0.6433 using a two-tailed unpaired t test). ( d ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) or vehicle (DMSO) before 10 μM isoproterenol treatment. ( e ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( f ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP (green), and CLC-dsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before before 10 μM isoproterenol treatment. ( g ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR or FLAG–β2AR-V2R when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( a, d, f ) show representative images from 3 independent experiments. ( b, e, g ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m.

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Phosphoinositide binding is essential for catalytic activation of β-arrestin trafficking but is dispensable for trafficking mediated by the scaffold mechanism ( a ) Live cell microscopy images of HEK 293 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP CCS mutant (green), and clathrin-light-chain–DsRed (red) before and after with 10 μM isoproterenol treatment. ( b ) Normalized plasma membrane (PM) fluorescence of β-arrestin-2–GFP lipid mutant in cells co-expressing FLAG–β2AR-V2R (n=12 cells from 3 independent experiments) when treated with 10 μM isoproterenol. ( c ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells expressing indicated β-arrestin-2–GFP construct before and after activation of FLAG-β2AR-V2R C tail with 10 μM isoproterenol (n=10, 12 cells, respectively, from 3 independent experiments; p=0.6433 using a two-tailed unpaired t test). ( d ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR (blue), β-arrestin-2-GFP (green), and clathrin-light-chain–DsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) or vehicle (DMSO) before 10 μM isoproterenol treatment. ( e ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( f ) Live cell microscopy images of COS-1 cells co-expressing FLAG–β2AR-V2R C tail (blue), β-arrestin-2-GFP (green), and CLC-dsRed (red) that have been pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before before 10 μM isoproterenol treatment. ( g ) Normalized average fold over initial β-arrestin-2–GFP fluorescence in cells co-expressing FLAG–β2AR or FLAG–β2AR-V2R when pre-treated for 1 hour with 1 μM phenylarsine oxide (PAO) before 10 μM isoproterenol treatment (n=12 cells from 3 independent experiments). ( a, d, f ) show representative images from 3 independent experiments. ( b, e, g ) show data as mean ± s.e.m. Scatter plots show overlay of mean and s.e.m.

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Binding Assay, Activation Assay, Microscopy, Expressing, Mutagenesis, Fluorescence, Construct, Two Tailed Test

    β-arrestin trafficking activation requires the GPCR core but not the GPCR cytoplasmic tail ( a ) Live cell TIRF microscopy images of COS-1 cells co-expressing FLAG–β2AR truncated at the 341 st amino acid (341T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells treated with 10 μM isoproterenol and co-expressing the indicated FLAG–β2AR (n=11 cells from 3 independent experiments, p=0.5634 using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–DRD2 G protein biased mutant (G prot, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( d ) Average (data shown as mean ± s.e.m.) and ( e ) maximum enrichment of β-arrestin-2–GFP into CCSs in cells expressing wild-type (green) or G protein biased mutant versions (gray) of FLAG-DRD2 and treated with 10 μM quinpirole (n=11 (WT) and 14 (G prot) cells from 3 independent experiments, p=0.013 using a two-tailed unpaired t test using Welch’s correction). ( a ) and ( c) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: β-arrestin trafficking activation requires the GPCR core but not the GPCR cytoplasmic tail ( a ) Live cell TIRF microscopy images of COS-1 cells co-expressing FLAG–β2AR truncated at the 341 st amino acid (341T, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( b ) Maximum β-arrestin-2–GFP enrichment at CCSs in cells treated with 10 μM isoproterenol and co-expressing the indicated FLAG–β2AR (n=11 cells from 3 independent experiments, p=0.5634 using a two-tailed unpaired t test). ( c ) Live cell TIRF microscopy images showing FLAG–DRD2 G protein biased mutant (G prot, blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM quinpirole treatment. ( d ) Average (data shown as mean ± s.e.m.) and ( e ) maximum enrichment of β-arrestin-2–GFP into CCSs in cells expressing wild-type (green) or G protein biased mutant versions (gray) of FLAG-DRD2 and treated with 10 μM quinpirole (n=11 (WT) and 14 (G prot) cells from 3 independent experiments, p=0.013 using a two-tailed unpaired t test using Welch’s correction). ( a ) and ( c) show representative images from 3 independent experiments. Scatter plots show overlay of mean and s.e.m. Scale bars, 5 μm. * p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Microscopy, Expressing, Two Tailed Test, Mutagenesis

    Differences in the bioenergetics of catalytic versus scaffold mechanisms of regulated β-arrestin trafficking and β-arrestin-dependent activation of ERK1/2 promoted by catalytic activation ( a ) Schematic depicting the proposed co-existence of catalytic and scaffolding mechanisms of β-arrestin trafficking tuned according to tail binding affinity, emphasizing the difference in tail versus core interactions (shaded boxes). The tail interaction, requiring GPCR phosphorylation (Rp) drives the scaffold mechanism through its essential role in stable GPCR/β-arrestin complex formation. The core interaction mediates catalysis by providing a kinetically favorable path for β-arrestin to remain captured at the PM irrespective of GPCR dissociation. Such capture requires phosphoinositide binding to the β-arrestin C-domain, explaining why the phosphoinositide requirement is specific to the catalytic mechanism and can be overcome by formation of a sufficiently sufficient stable scaffold complex requiring the phosphorylated GPCR tail. Primary energy inputs maintaining each proposed trafficking cycle are indicated by red arrows. The present results identify a specific requirement of the catalytic mechanism for phosphoinositide binding to the C-domain but they do not exclude binding also in the scaffold complex (which we think is likely). We also cannot presently rule out the possible existence of additional interaction(s) in the catalytic mechanism, such as phosphoinositide binding also to the β-arrestin N-domain that has the potential to displace the β-arrestin C-terminus 24 . ( b ) Representative images (from 3 independent experiments) before and after 10 μM isoproterenol treatment of cells expressing chimeric FLAG-tagged β1AR-V2Rs and imaged live with TIRF microscopy. Profiles of FLAG-β2AR and β-arrestin-2–GFP average enrichment into CCSs in COS-1 cells expressing either an empty vector construct ( c ) or GRK2 ( d ) and treated with 10 μM isoproterenol (n=15 or 12 cells, respectively, from 3 independent experiments). ( e ) Difference in enrichment values between β-arrestin-2–GFP and β2AR from panels c and d showing the effect of GRK2 overexpression. ( f ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β1AR and exposed to 10 μM isoproterenol for the indicated time period. ( g ) Quantification of ERK1/2 activation from the western blots in panel a (n=5 independent experiments, p=0.004 using a one-way ANOVA). ( h ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β2AR and exposed to 10 μM isoproterenol for the indicated time period. ( i ) Quantification of ERK1/2 activation from the western blots in panel c (n=5 independent experiments). ( f ) and ( h ) show representative Western blots from 5 independent experiments. Data shown as mean ± s.e.m. For gel source data, see Supplementary Figure 1 . Error bars represent s.e.m. ** p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Differences in the bioenergetics of catalytic versus scaffold mechanisms of regulated β-arrestin trafficking and β-arrestin-dependent activation of ERK1/2 promoted by catalytic activation ( a ) Schematic depicting the proposed co-existence of catalytic and scaffolding mechanisms of β-arrestin trafficking tuned according to tail binding affinity, emphasizing the difference in tail versus core interactions (shaded boxes). The tail interaction, requiring GPCR phosphorylation (Rp) drives the scaffold mechanism through its essential role in stable GPCR/β-arrestin complex formation. The core interaction mediates catalysis by providing a kinetically favorable path for β-arrestin to remain captured at the PM irrespective of GPCR dissociation. Such capture requires phosphoinositide binding to the β-arrestin C-domain, explaining why the phosphoinositide requirement is specific to the catalytic mechanism and can be overcome by formation of a sufficiently sufficient stable scaffold complex requiring the phosphorylated GPCR tail. Primary energy inputs maintaining each proposed trafficking cycle are indicated by red arrows. The present results identify a specific requirement of the catalytic mechanism for phosphoinositide binding to the C-domain but they do not exclude binding also in the scaffold complex (which we think is likely). We also cannot presently rule out the possible existence of additional interaction(s) in the catalytic mechanism, such as phosphoinositide binding also to the β-arrestin N-domain that has the potential to displace the β-arrestin C-terminus 24 . ( b ) Representative images (from 3 independent experiments) before and after 10 μM isoproterenol treatment of cells expressing chimeric FLAG-tagged β1AR-V2Rs and imaged live with TIRF microscopy. Profiles of FLAG-β2AR and β-arrestin-2–GFP average enrichment into CCSs in COS-1 cells expressing either an empty vector construct ( c ) or GRK2 ( d ) and treated with 10 μM isoproterenol (n=15 or 12 cells, respectively, from 3 independent experiments). ( e ) Difference in enrichment values between β-arrestin-2–GFP and β2AR from panels c and d showing the effect of GRK2 overexpression. ( f ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β1AR and exposed to 10 μM isoproterenol for the indicated time period. ( g ) Quantification of ERK1/2 activation from the western blots in panel a (n=5 independent experiments, p=0.004 using a one-way ANOVA). ( h ) Representative western blot showing phosphorylated ERK1/2 and total ERK1/2 signal in extracts prepared from parental or β-arrestin knockout CRISPR HEK 293 cells expressing FLAG–β2AR and exposed to 10 μM isoproterenol for the indicated time period. ( i ) Quantification of ERK1/2 activation from the western blots in panel c (n=5 independent experiments). ( f ) and ( h ) show representative Western blots from 5 independent experiments. Data shown as mean ± s.e.m. For gel source data, see Supplementary Figure 1 . Error bars represent s.e.m. ** p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Scaffolding, Binding Assay, Expressing, Microscopy, Plasmid Preparation, Construct, Over Expression, Western Blot, Knock-Out, CRISPR

    Verification of GPCR-specificity of the discrete β-arrestin trafficking mechanism, demonstration that this mechanism produces super-stoichiometric β-arrestin accumulation in CCSs and that its activation does not require the GPCR tail ( a ) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β1AR after the following treatments: 10 μM isoproterenol (green, n=14 cells), 15 minute pretreatment with 10 μM CGP 20712A and 10 μM isoproterenol treatment (red, n=12 cells), 10 μM CGP 20712A alone (gray, n=12 cells). Data shown for the 10 μM isoproterenol condition are replotted from Figure 1b . (b) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells transfected with the indicated receptor or empty vector and treated with 10 μM isoproterenol. (c) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated with 10 μM isoproterenol or 10 μM dobutamine (n=5 or 4 cells, respectively, from 2 independent experiments). (d) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated as indicated (n=12 cells). ( e ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β1AR (blue), β-arrestin-1–mVenus (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( f ) Enrichment into CCSs (n=7 cells from 3 independent experiments). (g) Maximum β-arrestin-1–mVenus enrichment at CCSs in HEK 293 cells transfected with FLAG-β1AR or empty vector and treated with 10 μM isoproterenol (n=7 and 11 cells from 3 independent experiments, p=0.0023 using an unpaired t test with Welch’s correction). (h) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β2AR after the following treatments: 10 μM isoproterenol (green, n=15 cells), 15 minute pretreatment with 10 μM ICI 118,551 and then 10 μM isoproterenol treatment (red, n=14 cells), 10 μM μM ICI 118,551 (gray, n=12). Data shown for the 10 μM isoproterenol condition are replotted from Figure 1d . ( i ) Fluorescence intensity profiles from lines shown in Figure 1e . ( j ) Time-dependent correlation coefficient of line scans across cells derived from immobilization experiments shown in Figure 1g, h ; n=3). ( k ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β2AR-GFP and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Fluorescence from the Alexa647 conjugated FLAG antibody shown in blue and GFP fluorescence shown in green. ( l ) Difference in GFP and Alexa647 fluorescence enrichment at CCSs in cells co-expressing FLAG-β1ARs (red), FLAG-β2ARs (blue) and β-arrestin-2-GFP or FLAG-β2AR-GFP (black). Cells were labeled with Alexa647 conjugated FLAG antibody for 10 minutes prior to live cell imaging. Data were derived from the experiments shown in Figure 1a, b (blue line, n=14 cells from 3 independent experiments), Figure 1c, d (red line, n=15 cells from 3 independent experiments), and Extended Data Figure 1k (black line n=12 cells from 3 independent experiments). ( m ) Plot of β-arrestin/GPCR stoichiometry calculated from the data displayed in panel k, calibrated according to the doubly labeled FLAG-β2AR-GFP reference construct defining 1:1 stoichiometry (For β1AR and β2AR, n=14 and 15 cells, respectively, from 3 independent experiments). A correction index was calculated by dividing GFP fluorescence by Alexa647 (FLAG) fluorescence in CCSs. This correction index was then applied to receptor and β-arrestin-2 enrichment in CCSs to determine β-arrestin-2/GPCR stoichiometry throughout the time course. Images were captured continuously at 0.5 Hz and stoichiometry values over the time course were calculated using a rolling average with 50-frame window size. Scale bar, 5 μm. Scatter plots show overlay of mean and s.e.m. ( a, d, h, j, l) show data as mean ± s.e.m. ** p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Verification of GPCR-specificity of the discrete β-arrestin trafficking mechanism, demonstration that this mechanism produces super-stoichiometric β-arrestin accumulation in CCSs and that its activation does not require the GPCR tail ( a ) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β1AR after the following treatments: 10 μM isoproterenol (green, n=14 cells), 15 minute pretreatment with 10 μM CGP 20712A and 10 μM isoproterenol treatment (red, n=12 cells), 10 μM CGP 20712A alone (gray, n=12 cells). Data shown for the 10 μM isoproterenol condition are replotted from Figure 1b . (b) Maximum β-arrestin-2–GFP enrichment at CCSs in HEK 293 cells transfected with the indicated receptor or empty vector and treated with 10 μM isoproterenol. (c) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated with 10 μM isoproterenol or 10 μM dobutamine (n=5 or 4 cells, respectively, from 2 independent experiments). (d) β-arrestin-2–GFP enrichment at CCSs in H9c2 cells without GPCR overexpression and treated as indicated (n=12 cells). ( e ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β1AR (blue), β-arrestin-1–mVenus (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. ( f ) Enrichment into CCSs (n=7 cells from 3 independent experiments). (g) Maximum β-arrestin-1–mVenus enrichment at CCSs in HEK 293 cells transfected with FLAG-β1AR or empty vector and treated with 10 μM isoproterenol (n=7 and 11 cells from 3 independent experiments, p=0.0023 using an unpaired t test with Welch’s correction). (h) Average β-arrestin-2–GFP enrichment at CCSs in cells expressing FLAG-β2AR after the following treatments: 10 μM isoproterenol (green, n=15 cells), 15 minute pretreatment with 10 μM ICI 118,551 and then 10 μM isoproterenol treatment (red, n=14 cells), 10 μM μM ICI 118,551 (gray, n=12). Data shown for the 10 μM isoproterenol condition are replotted from Figure 1d . ( i ) Fluorescence intensity profiles from lines shown in Figure 1e . ( j ) Time-dependent correlation coefficient of line scans across cells derived from immobilization experiments shown in Figure 1g, h ; n=3). ( k ) Live cell TIRF microscopy images (representative of n=3 independent experiments) showing FLAG–β2AR-GFP and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Fluorescence from the Alexa647 conjugated FLAG antibody shown in blue and GFP fluorescence shown in green. ( l ) Difference in GFP and Alexa647 fluorescence enrichment at CCSs in cells co-expressing FLAG-β1ARs (red), FLAG-β2ARs (blue) and β-arrestin-2-GFP or FLAG-β2AR-GFP (black). Cells were labeled with Alexa647 conjugated FLAG antibody for 10 minutes prior to live cell imaging. Data were derived from the experiments shown in Figure 1a, b (blue line, n=14 cells from 3 independent experiments), Figure 1c, d (red line, n=15 cells from 3 independent experiments), and Extended Data Figure 1k (black line n=12 cells from 3 independent experiments). ( m ) Plot of β-arrestin/GPCR stoichiometry calculated from the data displayed in panel k, calibrated according to the doubly labeled FLAG-β2AR-GFP reference construct defining 1:1 stoichiometry (For β1AR and β2AR, n=14 and 15 cells, respectively, from 3 independent experiments). A correction index was calculated by dividing GFP fluorescence by Alexa647 (FLAG) fluorescence in CCSs. This correction index was then applied to receptor and β-arrestin-2 enrichment in CCSs to determine β-arrestin-2/GPCR stoichiometry throughout the time course. Images were captured continuously at 0.5 Hz and stoichiometry values over the time course were calculated using a rolling average with 50-frame window size. Scale bar, 5 μm. Scatter plots show overlay of mean and s.e.m. ( a, d, h, j, l) show data as mean ± s.e.m. ** p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Over Expression, Microscopy, Fluorescence, Derivative Assay, Labeling, Live Cell Imaging, Construct

    sptPALM controls and mean square displacement (MSD) plots and cellular model (a) Representative image of a clathrin mask (green) generated from a CLC-GFP image (red). Representative diffusion maps overlaid with the clathrin mask for HEK 293 cells and treated with 10 μM isoproterenol expressing ( b ) PAmCherry-β1AR, ( c ) PAmCherry-β2AR, ( d ) β-arrestin-2-PAmCherry coexpressed with FLAG-β1AR, ( e ) β-arrestin-2-PAmCherry coexpressed with FLAG-β2AR ( f ) Distribution of diffusion coefficients (D) of false positive detections from HEK 293 cells expressing FLAG-β2AR and imaged under standard sptPALM acquisition conditions to determine contribution of false positive detections in the experimental setup and analysis. ( g ) Distribution of diffusion coefficients (D) of PAmCherry-β2AR, PAmCherry-PLCδ1-PH, and β-arrestin-2-PAmCherry in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13, 21, and 8 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. ( h ) Average MSD plots derived from sptPALM analysis of PAmCherry-β1AR and PAmCherry-β2AR trajectories in HEK 293 cells treated with 10 μM isoproterenol (n=8 and 13 cells, respectively). ( i ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β1AR in live HEK 293 cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13 and 17 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. ( j ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in cells co-expressing FLAG-β1AR and treated with 10 μM isoproterenol (n=13 and 17 cells, respectively). ( k ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β2AR in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=21 and 10 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel d. ( l ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in HEK 293 cells co-expressing FLAG-β2AR and treated with 10 μM isoproterenol (n=21 and 10 cells, respectively). ( m ) Immobile and ( n) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β1AR after 10 μM isoproterenol treatment. ( o ) Immobile and ( p) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β2AR after 10 μM isoproterenol treatment. Trajectory detections are false colored based on the density of detections at each pixel. Error bars represent s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images. ( q ) Proposed cellular pathway for catalytic activation of β-arrestin. ( r ) Representative microscopy images of COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) that were treated with 10 μM isoproterenol for 3 minutes. Then, β-arrestin-2-GFP was photobleached in the indicated yellow region (shown in inset; insets are also shown in Figure 5h ). ( a, b, c, d, e, m, n, o, p, and r ) show representative examples from at least 3 independent experiments. ( f–l ) show data as mean ± s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images; 5 μm for FRAP larger images and 0.5 μm for the insets.

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: sptPALM controls and mean square displacement (MSD) plots and cellular model (a) Representative image of a clathrin mask (green) generated from a CLC-GFP image (red). Representative diffusion maps overlaid with the clathrin mask for HEK 293 cells and treated with 10 μM isoproterenol expressing ( b ) PAmCherry-β1AR, ( c ) PAmCherry-β2AR, ( d ) β-arrestin-2-PAmCherry coexpressed with FLAG-β1AR, ( e ) β-arrestin-2-PAmCherry coexpressed with FLAG-β2AR ( f ) Distribution of diffusion coefficients (D) of false positive detections from HEK 293 cells expressing FLAG-β2AR and imaged under standard sptPALM acquisition conditions to determine contribution of false positive detections in the experimental setup and analysis. ( g ) Distribution of diffusion coefficients (D) of PAmCherry-β2AR, PAmCherry-PLCδ1-PH, and β-arrestin-2-PAmCherry in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13, 21, and 8 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry and PAmCherry-PLCδ1-PH were co-expressed individually with FLAG-β2AR. ( h ) Average MSD plots derived from sptPALM analysis of PAmCherry-β1AR and PAmCherry-β2AR trajectories in HEK 293 cells treated with 10 μM isoproterenol (n=8 and 13 cells, respectively). ( i ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β1AR in live HEK 293 cells imaged at 37°C after treatment with 10 μM isoproterenol (n=13 and 17 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. ( j ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in cells co-expressing FLAG-β1AR and treated with 10 μM isoproterenol (n=13 and 17 cells, respectively). ( k ) Distribution of diffusion coefficients (D) of β-arrestin-2-PAmCherry wild-type and CCS mutant when co-expressed with FLAG-β2AR in live cells imaged at 37°C after treatment with 10 μM isoproterenol (n=21 and 10 cells, respectively). Black lines show diffusion coefficient profiles that have not been corrected for false positive detections, showing limited contribution to the profiles. β-arrestin-2-PAmCherry diffusion coefficient profiles when activated by the β2AR are replotted from panel d. ( l ) Average MSD plots derived from sptPALM analysis of β-arrestin-2-PAmCherry wild-type and CCS mutant trajectories in HEK 293 cells co-expressing FLAG-β2AR and treated with 10 μM isoproterenol (n=21 and 10 cells, respectively). ( m ) Immobile and ( n) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β1AR after 10 μM isoproterenol treatment. ( o ) Immobile and ( p) mobile β-arrestin-2-PAmCherry trajectory detections overlaid with a clathrin marker (red) in live cells co-expressing FLAG-β2AR after 10 μM isoproterenol treatment. Trajectory detections are false colored based on the density of detections at each pixel. Error bars represent s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images. ( q ) Proposed cellular pathway for catalytic activation of β-arrestin. ( r ) Representative microscopy images of COS-1 cells co-expressing FLAG-β2AR, β-arrestin-2-GFP (green), and clathrin-light-chain-DsRed (red) that were treated with 10 μM isoproterenol for 3 minutes. Then, β-arrestin-2-GFP was photobleached in the indicated yellow region (shown in inset; insets are also shown in Figure 5h ). ( a, b, c, d, e, m, n, o, p, and r ) show representative examples from at least 3 independent experiments. ( f–l ) show data as mean ± s.e.m; in some cases, error bars are smaller than the height of the symbol and, therefore, not shown. Scale bars, 500 nm for sptPALM images; 5 μm for FRAP larger images and 0.5 μm for the insets.

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Generated, Diffusion-based Assay, Expressing, Derivative Assay, Mutagenesis, Marker, Activation Assay, Microscopy

    Discrete mode of GPCR-activated cellular β-arrestin trafficking is broadly conserved ( a–d ) Live cell TIRF microscopy images showing ( a) FLAG–β1AR (blue) or (c) FLAG–β2AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Average enrichment at CCSs after 10 μM isoproterenol treatment for ( b) FLAG–β1AR ( d ) FLAG–β2AR (n=14 and 15 cells, respectively, from 3 independent experiments, data shown as mean ± s.e.m.). ( e ) Live cell TIRF microscopy images of HEK 293 cells co-expressing super ecliptic pHluorin–β2AR (blue), β-arrestin-2–mApple (green), and clathrin-light-chain–TagBFP (red) before and after 10 μM isoproterenol treatment. ( f ) Timelapse of individual pre-existing CCSs from panel e. Scale bars, 5 μm. ( a, c, e, f) show representative images from 3 independent experiments.

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Discrete mode of GPCR-activated cellular β-arrestin trafficking is broadly conserved ( a–d ) Live cell TIRF microscopy images showing ( a) FLAG–β1AR (blue) or (c) FLAG–β2AR (blue), β-arrestin-2–GFP (green) and clathrin-light-chain–DsRed (red) before and after 10 μM isoproterenol treatment. Average enrichment at CCSs after 10 μM isoproterenol treatment for ( b) FLAG–β1AR ( d ) FLAG–β2AR (n=14 and 15 cells, respectively, from 3 independent experiments, data shown as mean ± s.e.m.). ( e ) Live cell TIRF microscopy images of HEK 293 cells co-expressing super ecliptic pHluorin–β2AR (blue), β-arrestin-2–mApple (green), and clathrin-light-chain–TagBFP (red) before and after 10 μM isoproterenol treatment. ( f ) Timelapse of individual pre-existing CCSs from panel e. Scale bars, 5 μm. ( a, c, e, f) show representative images from 3 independent experiments.

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Microscopy, Expressing

    Phosphoinositide binding is required to capture β-arrestin at the plasma membrane after GPCR dissociation Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR, the indicated β-arrestin-2–GFP construct, and clathrin-light-chain–DsRed. Shown are β-arrestin images false-colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (shown as mean ± s.e.m), respectively, from cells co-expressing FLAG–β2ARs without isoproterenol treatment ( a–c ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( d–f ), lipid mutant ( g–i ), CCS mutant ( j–l ), and CCS and lipid mutant ( m–o ); n=12 cells per condition. Wild-type β-arrestin-2–GFP maximum enrichment in panel h is replotted from panel e and panel n is replotted from panel k. Statistics were calculated using a two-tailed unpaired t test with Welch’s correction. For ( h ) n=12 and 11 cells, respectively, from 3 independent experiments and p=0.0006. For ( k ) n=10 cells from 3 independent experiments and p=0.0102. For ( n ) n=10 cells from 3 independent experiments and p=0.0022. Extended Data Table 1 provides detailed description of β-arrestin mutations. Scatter plots show overlay of mean and s.e.m. scale bars, 5 μm. ** p

    Journal: Nature

    Article Title: Catalytic activation of β-arrestin by GPCRs

    doi: 10.1038/s41586-018-0079-1

    Figure Lengend Snippet: Phosphoinositide binding is required to capture β-arrestin at the plasma membrane after GPCR dissociation Representative live cell TIRF microscopy images (from 3 independent experiments) showing FLAG–β2AR, the indicated β-arrestin-2–GFP construct, and clathrin-light-chain–DsRed. Shown are β-arrestin images false-colored to indicate fluorescence intensity, maximum fluorescence enrichment at CCSs, and normalized average plasma membrane (PM) β-arrestin-2–GFP fluorescence (shown as mean ± s.e.m), respectively, from cells co-expressing FLAG–β2ARs without isoproterenol treatment ( a–c ), and the following β-arrestin-2–GFP constructs with 10 μM isoproterenol treatment: wild-type ( d–f ), lipid mutant ( g–i ), CCS mutant ( j–l ), and CCS and lipid mutant ( m–o ); n=12 cells per condition. Wild-type β-arrestin-2–GFP maximum enrichment in panel h is replotted from panel e and panel n is replotted from panel k. Statistics were calculated using a two-tailed unpaired t test with Welch’s correction. For ( h ) n=12 and 11 cells, respectively, from 3 independent experiments and p=0.0006. For ( k ) n=10 cells from 3 independent experiments and p=0.0102. For ( n ) n=10 cells from 3 independent experiments and p=0.0022. Extended Data Table 1 provides detailed description of β-arrestin mutations. Scatter plots show overlay of mean and s.e.m. scale bars, 5 μm. ** p

    Article Snippet: Samples were then separated by SDS–PAGE (Life Technologies) and transferred to a nitrocellulose membrane that was blocked with TBS Odyssey blocking buffer (LI-COR) for one hour at room temperature and then incubated overnight at 4 °C with a mouse anti-adaptin β primary antibody (1:250, BD Biosciences 610382) and a rabbit anti-GFP primary antibody (1:500, Thermo Fisher Scientific A-11122).

    Techniques: Binding Assay, Microscopy, Construct, Fluorescence, Expressing, Mutagenesis, Two Tailed Test

    Chronic hypoxia and pulmonary HIMF gene transfer increase the number of BMD cells associated with the pulmonary vasculature. A–D: Paraffin-embedded lung sections from mice exposed to normoxia (7d, 20.8% O 2 ) (A), hypoxia (7d, 10.0% O 2 ) (B), AAV-null (14d, 2.5×10 10 VP) (C), or AAV-HIMF (14d, 2.5×10 10 VP) (D) were probed with polyclonal antibodies raised against GFP. Arrows indicate GFP + cells within the vasculature. Scale bar: 50 µm. E, F: Quantification of GFP + cells within the pulmonary vasculature. GFP + cells within the pulmonary vasculature are shown as mean ± SEM of GFP + cells/vessel. * P

    Journal: PLoS ONE

    Article Title: Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELM?) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

    doi: 10.1371/journal.pone.0011251

    Figure Lengend Snippet: Chronic hypoxia and pulmonary HIMF gene transfer increase the number of BMD cells associated with the pulmonary vasculature. A–D: Paraffin-embedded lung sections from mice exposed to normoxia (7d, 20.8% O 2 ) (A), hypoxia (7d, 10.0% O 2 ) (B), AAV-null (14d, 2.5×10 10 VP) (C), or AAV-HIMF (14d, 2.5×10 10 VP) (D) were probed with polyclonal antibodies raised against GFP. Arrows indicate GFP + cells within the vasculature. Scale bar: 50 µm. E, F: Quantification of GFP + cells within the pulmonary vasculature. GFP + cells within the pulmonary vasculature are shown as mean ± SEM of GFP + cells/vessel. * P

    Article Snippet: Polyclonal rabbit anti-GFP antibodies were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Mouse Assay

    Both chronic hypoxia and pulmonary HIMF gene transfer recruit BMD cells to the pulmonary vasculature. (A–C, J–M) Light micrograph of fluorescence images to show blood vessel structure. Frozen sections from normoxic (20.8% O 2 ) (D, N), hypoxic (10.0% O 2 ) (E, O), and AAV-HIMF treated (2.5×10 10 VP) (F, P, Q) lungs were stained with a rabbit anti-GFP polyclonal antibody that was visualized by an FITC-conjugated goat anti-rabbit IgG antibody (green). (G–I, R–U): Differential interference contrast images of light and fluorescence images to show structure. A–L, N–P, R–T: Scale bar: 50µm. M, Q, U: Scale bar: 20µm.

    Journal: PLoS ONE

    Article Title: Hypoxia-Induced Mitogenic Factor (HIMF/FIZZ1/RELM?) Recruits Bone Marrow-Derived Cells to the Murine Pulmonary Vasculature

    doi: 10.1371/journal.pone.0011251

    Figure Lengend Snippet: Both chronic hypoxia and pulmonary HIMF gene transfer recruit BMD cells to the pulmonary vasculature. (A–C, J–M) Light micrograph of fluorescence images to show blood vessel structure. Frozen sections from normoxic (20.8% O 2 ) (D, N), hypoxic (10.0% O 2 ) (E, O), and AAV-HIMF treated (2.5×10 10 VP) (F, P, Q) lungs were stained with a rabbit anti-GFP polyclonal antibody that was visualized by an FITC-conjugated goat anti-rabbit IgG antibody (green). (G–I, R–U): Differential interference contrast images of light and fluorescence images to show structure. A–L, N–P, R–T: Scale bar: 50µm. M, Q, U: Scale bar: 20µm.

    Article Snippet: Polyclonal rabbit anti-GFP antibodies were purchased from Invitrogen (Carlsbad, CA).

    Techniques: Fluorescence, Staining

    Lead triggers phase separation of TDP-43 and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p

    Journal: Toxicological Sciences

    Article Title: Heavy Metal Neurotoxicants Induce ALS-Linked TDP-43 Pathology

    doi: 10.1093/toxsci/kfy267

    Figure Lengend Snippet: Lead triggers phase separation of TDP-43 and decreases its solubility. A, Purified TDP-43 LLPS in vitro is facilitated by lead (II) acetate trihydrate (Pb) in a dose-dependent manner. Representative 63× DIC images. Arrows: examples of single TDP-43 droplets. Asterisk: examples of amorphous TDP-43 consolidates. Arrowheads: high-contrast, inert 1 micron polystyrene microspheres added to aid sample focusing. Scale bar = 10 µm. B, LLPS was quantified using an ImageJ algorithm as percentage of ROI covered by droplets. Points at mean, with error bars at SEM, were fit by nonlinear regression analysis (line) and the LogEC50 calculated as 160 µM (dotted line). Induced PC12 cells accumulate insoluble TDP-43:: GFP upon treatment with lead (Pb). Immunoblots of RIPA insoluble (C), RIPA soluble (D) and total RIPA lysate (E) were probed with anti-GFP antibodies ( N = 3). F, Densitometric analysis of total TDP-43:: GFP/Actin bands (from panel E) show an increase in TDP-43:: GFP in response to 0.174 and 0.521 µM lead but an decrease in response to 4.69 µM lead. G, At 1.56 and 4.69 µM concentrations, lead significantly increases the ratio of insoluble TDP-43:: GFP to soluble TDP-43:: GFP. N = 3; mean ± SEM; ANOVA w/Dunnett’s multiple comparison test, * p

    Article Snippet: Blots were incubated with the following antibodies: 1:1000 anti-phospho-S409/410 TDP-43 (a gift from Leonard Petrucelli, Mayo Clinic, Rb3655), 1:2000 anti-GFP (Sigma; G1544), 1:2000 anti-TDP-43 (ProteinTech; 12892–1-AP); and 1:10000 anti-Actin (Millipore; MAB1501).

    Techniques: Solubility, Purification, In Vitro, Western Blot

    Physical interactions between dTgs1 and the SMN complex. Proteins co-purifying with dTgs1 or Smn were identified by affinity purification (AP) using GFP-TRAP beads, followed by mass spectrometry (MS) and stringent filtering (see Materials and Methods for details). AP/MS was carried out using 0–3 hr embryos from mothers expressing UAS-GFP-dTgs1 driven by Actin-Gal4 or Smn-GFP under the control of the tubulin promoter. (A) Efficiency of GFP-TRAP-mediated AP assayed using an anti-GFP antibody (T, total protein extract; I, input (10%); S supernatant; IP, immunoprecipitate). (B) Schematic representations of the snRNP maturation pathway; note that in Drosophila there are only four Gemin proteins. See Introduction for a detailed description of this pathway. (C, D) dTgs1 (C) and Smn (D) interacting proteins. All protein IDs conforming to stringent filtering (see Materials and Methods ) are shown in the Tables. Mean area corresponds to top 3 protein quantification (T3PQ), the mean of the three highest abundance peptides identified for each protein. The complete lists of the Tgs1- and Smn-interacting proteins are shown in S1 and S2 Tables.

    Journal: PLoS Genetics

    Article Title: Intimate functional interactions between TGS1 and the Smn complex revealed by an analysis of the Drosophila eye development

    doi: 10.1371/journal.pgen.1008815

    Figure Lengend Snippet: Physical interactions between dTgs1 and the SMN complex. Proteins co-purifying with dTgs1 or Smn were identified by affinity purification (AP) using GFP-TRAP beads, followed by mass spectrometry (MS) and stringent filtering (see Materials and Methods for details). AP/MS was carried out using 0–3 hr embryos from mothers expressing UAS-GFP-dTgs1 driven by Actin-Gal4 or Smn-GFP under the control of the tubulin promoter. (A) Efficiency of GFP-TRAP-mediated AP assayed using an anti-GFP antibody (T, total protein extract; I, input (10%); S supernatant; IP, immunoprecipitate). (B) Schematic representations of the snRNP maturation pathway; note that in Drosophila there are only four Gemin proteins. See Introduction for a detailed description of this pathway. (C, D) dTgs1 (C) and Smn (D) interacting proteins. All protein IDs conforming to stringent filtering (see Materials and Methods ) are shown in the Tables. Mean area corresponds to top 3 protein quantification (T3PQ), the mean of the three highest abundance peptides identified for each protein. The complete lists of the Tgs1- and Smn-interacting proteins are shown in S1 and S2 Tables.

    Article Snippet: Membranes were probed with rabbit anti-Tgs1 antiserum (1:5000; this study), mouse anti-tubulin (1:20000; Sigma-Aldrich), mouse anti-beta Actin (1:100000, Abcam 49900 [AC-15], HRP), rabbit anti-human TGS1 (1:1500, Bethyl Laboratories Cat#A300-814A, lot 1), rabbit anti-GFP (1:1000; Torrey Pines Biolabs, TP401) antibodies.

    Techniques: Affinity Purification, Mass Spectrometry, Expressing

    Diffusion of GFP takes a random route via the NPC. Representative TEM micrographs of high-pressure frozen and freeze-substituted yeast expressing unconjugated GFP immunolabelled with polyclonal anti-GFP primary antibody and anti-rabbit secondary antibody

    Journal: Journal of Cell Science

    Article Title: Facilitated transport and diffusion take distinct spatial routes through the nuclear pore complex

    doi: 10.1242/jcs.070730

    Figure Lengend Snippet: Diffusion of GFP takes a random route via the NPC. Representative TEM micrographs of high-pressure frozen and freeze-substituted yeast expressing unconjugated GFP immunolabelled with polyclonal anti-GFP primary antibody and anti-rabbit secondary antibody

    Article Snippet: Primary antibodies used in this study were rabbit polyclonal anti-GFP (Abcam, Cambridge, UK) used in 1:50 dilution, affinity-purified rabbit polyclonal antibody against Gle1 (anti-Gle1) [WU851 ( )] in 1:10 dilution and affinity-purified rabbit polyclonal antibody against Dbp5 (anti-Dbp5) [ASW42 ( )].

    Techniques: Diffusion-based Assay, Transmission Electron Microscopy, Expressing

    BDFN overexpression rescues spine phenotype due to eEF2K knock-down. A , Neurons were transfected with GFP, BDNF, sieEF2K, or sieEF2K+BDNF vectors on DIV10, fixed on DIV18, and stained with BDNF antibody. BDNF coexpression increased the BDNF signal along the dendrites and positively affected spine number and morphology in comparison with sieEF2K alone. B–D , Mean length, width and number of dendritic spines (±SEM) in neurons transfected with the indicated constructs; > 10 transfected neurons (corresponding to > 5000 μm in dendrite length), were measured for every condition; * p

    Journal: The Journal of Neuroscience

    Article Title: Synaptic Activity Controls Dendritic Spine Morphology by Modulating eEF2-Dependent BDNF Synthesis

    doi: 10.1523/JNEUROSCI.0119-10.2010

    Figure Lengend Snippet: BDFN overexpression rescues spine phenotype due to eEF2K knock-down. A , Neurons were transfected with GFP, BDNF, sieEF2K, or sieEF2K+BDNF vectors on DIV10, fixed on DIV18, and stained with BDNF antibody. BDNF coexpression increased the BDNF signal along the dendrites and positively affected spine number and morphology in comparison with sieEF2K alone. B–D , Mean length, width and number of dendritic spines (±SEM) in neurons transfected with the indicated constructs; > 10 transfected neurons (corresponding to > 5000 μm in dendrite length), were measured for every condition; * p

    Article Snippet: The following antibodies and dilutions were used (sources in parentheses): rabbit anti-P-eEF2 1:1000 and rabbit anti-eEF2 1:1000 (gifts from A. C. Nairn, Yale University, New Haven, CT); rabbit anti-CaMKII 1:1000, rabbit anti-eEF2 kinase 1:1000, rabbit anti-ERK 1/2 1:500, rabbit anti P-ERK 1/2 1:500, rabbit anti-S6K 1:500, rabbit anti-4eBP1 1:250 and rabbit anti-GFP 1:500 (Cell Signaling Technology); rabbit anti-BDNF 1:500, rabbit anti-ARC 1:200, anti-NGF 1:1000 (Santa Cruz Biotechnology); rabbit anti-GluR2 1:400 (NeuroMab, UC Davis/NIH NeuroMab Facility); rabbit anti pro-BDNF 1:1000, mouse anti-vimentin 1:1000, mouse anti-β-actin 1:1000, and mouse anti-α-tubulin 1:1000 (Sigma).

    Techniques: Over Expression, Transfection, Staining, Construct

    Transcription factor Brn3b promoted an increase in the levels of p-AKT in retinas of rats injected with rAAV-hSyn-Brn3b. A : Immunostaining for p-AKT (pseudogreen), βIII-tubulin (pseudored) expression in retinal sections from Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–hSyn–green fluorescent protein (rAAV-hSyn-GFP; vector control) or rAAV-hSyn-Brn3b virus. The immunostaining was detected using corresponding Alexa 546 (pseudogreen) or Alexa 647 (pseudored) conjugated secondary antibody. Scale bar indicates 20 µm. B : A magnified view of the retinal ganglion cell (RGC) layers of retinas transduced with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. C : A significant 2.4-fold increase in p-AKT expression was observed in the RGCs of rats injected with rAAV-hSyn-Brn3b. Ratios of fluorescence intensity values are shown in mean ± standard error of the mean (SEM), n = 6. The Mann–Whitney rank-sum test was used for statistical analysis (*p

    Journal: Molecular Vision

    Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

    doi:

    Figure Lengend Snippet: Transcription factor Brn3b promoted an increase in the levels of p-AKT in retinas of rats injected with rAAV-hSyn-Brn3b. A : Immunostaining for p-AKT (pseudogreen), βIII-tubulin (pseudored) expression in retinal sections from Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–hSyn–green fluorescent protein (rAAV-hSyn-GFP; vector control) or rAAV-hSyn-Brn3b virus. The immunostaining was detected using corresponding Alexa 546 (pseudogreen) or Alexa 647 (pseudored) conjugated secondary antibody. Scale bar indicates 20 µm. B : A magnified view of the retinal ganglion cell (RGC) layers of retinas transduced with either rAAV-hSyn-GFP or rAAV-hSyn-Brn3b. C : A significant 2.4-fold increase in p-AKT expression was observed in the RGCs of rats injected with rAAV-hSyn-Brn3b. Ratios of fluorescence intensity values are shown in mean ± standard error of the mean (SEM), n = 6. The Mann–Whitney rank-sum test was used for statistical analysis (*p

    Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

    Techniques: Injection, Immunostaining, Expressing, Recombinant, Plasmid Preparation, Transduction, Fluorescence, MANN-WHITNEY

    Levels of p-AKT in the retinas of rats with elevated IOP overexpressing Brn3b. A : Immunostaining for p-AKT in retinal ganglion cells (RGCs) of Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–cytomegalovirus–green fluorescent protein (rAAV-CMV-GFP) or rAAV-CMV-Brn3b following intraocular pressure (IOP) elevation. Retinal sections obtained were immunostained for p-AKT (pseudogreen) and βIII-tubulin (pseudored). B : An increase in immunostaining (not statistically significant) for p-AKT was observed in RGCs overexpressing Brn3b (rAAV-CMV-Brn3b) compared to RGCs overexpressing the control vector (rAAV-CMV-GFP; determined with the L/R ratios of fluorescence intensities of p-AKT staining in RGCs). Values are represented as mean ± standard error of the mean (SEM), n = 3. Scale bar indicates 20 µm.

    Journal: Molecular Vision

    Article Title: Bcl-2, Bcl-xL, and p-AKT are involved in neuroprotective effects of transcription factor Brn3b in an ocular hypertension rat model of glaucoma

    doi:

    Figure Lengend Snippet: Levels of p-AKT in the retinas of rats with elevated IOP overexpressing Brn3b. A : Immunostaining for p-AKT in retinal ganglion cells (RGCs) of Brown Norway rats intravitreally injected with either the recombinant adenoassociated virus–cytomegalovirus–green fluorescent protein (rAAV-CMV-GFP) or rAAV-CMV-Brn3b following intraocular pressure (IOP) elevation. Retinal sections obtained were immunostained for p-AKT (pseudogreen) and βIII-tubulin (pseudored). B : An increase in immunostaining (not statistically significant) for p-AKT was observed in RGCs overexpressing Brn3b (rAAV-CMV-Brn3b) compared to RGCs overexpressing the control vector (rAAV-CMV-GFP; determined with the L/R ratios of fluorescence intensities of p-AKT staining in RGCs). Values are represented as mean ± standard error of the mean (SEM), n = 3. Scale bar indicates 20 µm.

    Article Snippet: The sections were double-immunostained with mouse anti-βIII-tubulin antibody in combination with either rabbit anti-Brn3b antibody (1:250 dilution, Antibody Research Corporation, St. Charles, MO), rabbit anti-Bcl-2 antibody (1:100 dilution, catalog no. sc-492; Santa Cruz Technology, Dallas, TX), rabbit anti-Bcl-xL antibody (1:300 dilution, catalog no. 2764; Cell Signaling Technology, Beverly, MA), rabbit anti-p-AKT antibody (1:25 dilution, catalog no. 9271; Cell Signaling Technology), or rabbit anti-GFP antibody (1:100 dilution, catalog no. G10362; ThermoFisher, Waltham, MA) and incubated overnight at 4 °C.

    Techniques: Immunostaining, Injection, Recombinant, Plasmid Preparation, Fluorescence, Staining

    Mitochondrial dynamics in living cells analyzed using GFP photoactivation and time-lapse microscopy. HeLa cells were co-transfected with a mitochondrial matrix targeted photoactivable GFP (mito-PAGFP) and mRFP, or mRFP-Htt28Q or mRFP-Htt74Q expression plasmids. After 24 h transfection, cells were subjected to photoactivation studies, or lysates were prepared from the cells for immunoblot analysis. ( A ) Expression of mRFP, mRFP-Htt28Q and mRFP-Htt74Q proteins detected by immunnoblotting proteins lysates from the transfected cells with a rabbit polyclonal anti-RFP antibody. ( B ) Mitochondria in cells expressing mRFP or mRFP-Htt28Q proteins display more dynamic movement and fusion compared with cells expressing mRFP-Htt74Q protein. HeLa cells that had been co-transfected with mito-PAGFP and mRFP, or mito-PAGFP and mRFP-Htt28Q, or mito-PAGFP and mRFP-Htt74Q were imaged under a fluorescent microscope using a 100× objective lens and those displaying RFP fluorescence were targeted for photoactivation of the co-transfected mito-PAGFP protein by illumination with 405 nm light, and then GFP fluorescence images were captured over the indicated time intervals. Arrowheads point to the mitochondria that fused with one another in the time period shown. Please note that in the GFP-Htt74Q transfected cell, mitochondria were smaller, clustered, lacked dynamic movement and fusion events were very infrequent. ( C ) Same as in (B), except that the images shown were captured using a 40× objective. The left-hand panels show the mRFP fluorescence seen in a group of cells and the region (indicated by the circle) that was illuminated with 405 nm light to photoactivate the co-expressed PAGFP-mito protein in the various transfected cells. The subsequent GFP fluorescence images captured at 0, 15 and 30 min after GFP photoactivation are shown on the right of the RFP fluorescence image captured for each construct. ( D ) Quantification of the changes in GFP fluorescence intensity over time in cells transfected with the different mRFP-tagged expression constructs. GFP fluorescence was measured at 0, 15 and 30 min after photoactivation in the photoactivated (a, b and c) and non-activated regions (e, f and g, respectively) in cells transfected with mRFP or mRFP-Htt28Q or mRFP-Htt74Q expression constructs, respectively. The plots depict the results obtained in 10 independent experiments (each shown with a different color). Note that GFP fluorescence in the photoactivated regions, in general, decreases faster in the cells expressing either mRFP, or mRFP-Htt28Q proteins, compared with those expressing mRFP-Htt74Q protein. These changes were accompanied by a gradual increase in GFP fluorescent intensity in the non-activated regions of cells expressing either mRFP or mRFP-Htt28Q proteins, but remained relatively constant in cells expressing mRFP-Htt74Q. These results are consistent with the idea that mitochondria in cells expressing either the mRFP or the mRFP-HttQ28 proteins exhibit greater mitochondria fusion than cells expressing the mRFP-HttQ74 protein.

    Journal: Human Molecular Genetics

    Article Title: Effects of overexpression of Huntingtin proteins on mitochondrial integrity

    doi: 10.1093/hmg/ddn404

    Figure Lengend Snippet: Mitochondrial dynamics in living cells analyzed using GFP photoactivation and time-lapse microscopy. HeLa cells were co-transfected with a mitochondrial matrix targeted photoactivable GFP (mito-PAGFP) and mRFP, or mRFP-Htt28Q or mRFP-Htt74Q expression plasmids. After 24 h transfection, cells were subjected to photoactivation studies, or lysates were prepared from the cells for immunoblot analysis. ( A ) Expression of mRFP, mRFP-Htt28Q and mRFP-Htt74Q proteins detected by immunnoblotting proteins lysates from the transfected cells with a rabbit polyclonal anti-RFP antibody. ( B ) Mitochondria in cells expressing mRFP or mRFP-Htt28Q proteins display more dynamic movement and fusion compared with cells expressing mRFP-Htt74Q protein. HeLa cells that had been co-transfected with mito-PAGFP and mRFP, or mito-PAGFP and mRFP-Htt28Q, or mito-PAGFP and mRFP-Htt74Q were imaged under a fluorescent microscope using a 100× objective lens and those displaying RFP fluorescence were targeted for photoactivation of the co-transfected mito-PAGFP protein by illumination with 405 nm light, and then GFP fluorescence images were captured over the indicated time intervals. Arrowheads point to the mitochondria that fused with one another in the time period shown. Please note that in the GFP-Htt74Q transfected cell, mitochondria were smaller, clustered, lacked dynamic movement and fusion events were very infrequent. ( C ) Same as in (B), except that the images shown were captured using a 40× objective. The left-hand panels show the mRFP fluorescence seen in a group of cells and the region (indicated by the circle) that was illuminated with 405 nm light to photoactivate the co-expressed PAGFP-mito protein in the various transfected cells. The subsequent GFP fluorescence images captured at 0, 15 and 30 min after GFP photoactivation are shown on the right of the RFP fluorescence image captured for each construct. ( D ) Quantification of the changes in GFP fluorescence intensity over time in cells transfected with the different mRFP-tagged expression constructs. GFP fluorescence was measured at 0, 15 and 30 min after photoactivation in the photoactivated (a, b and c) and non-activated regions (e, f and g, respectively) in cells transfected with mRFP or mRFP-Htt28Q or mRFP-Htt74Q expression constructs, respectively. The plots depict the results obtained in 10 independent experiments (each shown with a different color). Note that GFP fluorescence in the photoactivated regions, in general, decreases faster in the cells expressing either mRFP, or mRFP-Htt28Q proteins, compared with those expressing mRFP-Htt74Q protein. These changes were accompanied by a gradual increase in GFP fluorescent intensity in the non-activated regions of cells expressing either mRFP or mRFP-Htt28Q proteins, but remained relatively constant in cells expressing mRFP-Htt74Q. These results are consistent with the idea that mitochondria in cells expressing either the mRFP or the mRFP-HttQ28 proteins exhibit greater mitochondria fusion than cells expressing the mRFP-HttQ74 protein.

    Article Snippet: The antibodies used for western blotting were a rabbit polyclonal anti-GFP (1:3000, generated to recombinant GST–GFP fusion protein by us), a rabbit polyclonal anti-RFP (1:3000, generated to recombinant GST–RFP fusion protein by us), a rabbit polyclonal anti-Mfn2 (1:1000), a rabbit polyclonal (sc-32898, Santa Cruz Biotechnology) and a mouse monoclonal anti-Drp1 (DLP-1; 1:1000, BD Biosciences), a mouse monoclonal anti-Mfn1 (clone 3C9, Novus Biologicals, CO, USA) or a goat polyclonal anti-actin (1:2000) antibody (Santa Cruz Biotechnology).

    Techniques: Time-lapse Microscopy, Transfection, Expressing, Microscopy, Fluorescence, Construct