polyclonal rabbit antibodies 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 polyclonal antibody
    Endogenous TRAPPC8 localizes to the centrosome/basal body. RPE cells were serum-starved for 24 h, fixed with methanol (upper two panels) or PFA (lower panel) and stained with rabbit <t>polyclonal</t> antibody against TRAPPC8 (green) and mouse monoclonal antibody against p150 Glued , rat monoclonal antibody against EB3 or mouse monoclonal antibody against acetylated tubulin (Ac tub), as indicated (red). DNA was stained with DAPI. Arrowheads and asterisks point to the centrosomes/basal bodies. Closed arrow indicates a primary cilium.
    Rabbit Polyclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7314 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Abcam rabbit polyclonal antibody
    The effect of ATRA treatment on expression of ANXA2 and S100A10 mRNA and protein levels. The effect on ANXA2 and S100A10 expression following 1–6 days ATRA treatment (1, 5 μM) in OAW28 ( a and c ) and OV-90 ( b and d ) cells. ANXA2 and S100A10 expression was assessed using 2 -∆∆CT quantitation method and normalised to housekeeping gene β-actin using no treatment control as a calibrator. Data is from 2 to 4 independent experiments ( n = 4–12). Statistical significance was determined using one-way ANOVA and the Tukey multiple comparison post hoc test. Western blots for annexin A2 and S100A10 ( e and f ) following 6 days ATRA treatment (1, 5 μM). Annexin A2 bands at 38 kDa, S100A10 at 11 kDa and β-actin bands at 42 kDa are shown for OAW28 ( e ) and OV-90 ( f ) cells. Equal amounts protein (5 μg for annexin A2 15 μg S100A10 were run on a 4–12% SDS-PAGE gel and immunoblotted with mouse monoclonal antibodies to annexin A2 (1/2000, BD Biosciences) or S100A10 (1/2000, BD Biosciences). A rabbit <t>polyclonal</t> antibody was used detect β-actin (1/2000, ab8227, Abcam). Numbers below western bands in e and f are fold changes relative to the control treatment. Westerns blots are representative of two independent experiments
    Rabbit Polyclonal Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 92/100, based on 11154 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology rabbit polyclonal antibody
    The requirements for the translocation of ICP0 from the nucleus to the cytoplasm. (A) ICP0 carrying the D199A substitution encoded by R7914 is not translocated to the cytoplasm late in infection of HEL fibroblasts. Replicate slide cultures of HEL fibroblasts were infected with 20 PFU of HSV-1(F), the recombinant virus R7914, or the repaired virus R7915 per cell and maintained at 37°C. At 12 h after infection, the cells were fixed and reacted first with <t>polyclonal</t> rabbit serum directed against exon II of ICP0 and second with FITC-conjugated goat anti-rabbit immunoglobulin antibodies. Sequential fields were examined in a Zeiss confocal microscope, and the numbers of cells exhibiting nuclear, cytoplasmic, or both nuclear and cytoplasmic localization of ICP0 were tabulated as shown in the histogram. The numbers above the bars indicate the numbers of cells showing a specific distribution of ICP0. (B) A viral gene function mediates the translocation of ICP0 from the nucleus to the cytoplasm during productive infection. Replicate slide cultures of HEL fibroblasts were exposed first to recombinant baculoviruses encoding wild-type or mutant ICP0 (D199A). After 2 h, cells were treated with 5 mM Na-butyrate. At 3 h after exposure to baculoviruses, the cells were exposed to the recombinant virus R7910, from which both copies of the α0 gene had been deleted. After 15 h of exposure to baculoviruses alone or 12 h after infection with R7910, the cells were fixed and reacted with rabbit polyclonal antibody against ICP0 and mouse monoclonal antibody against gD and then reacted with FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG antibodies. Cell counts were done as described above, except that only cells exhibiting gD encoded by R7910 and ICP0 encoded by baculoviruses were counted.
    Rabbit Polyclonal Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 10614 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc rabbit polyclonal antibody
    Lactosylceramide is associated with HCV NS4B protein. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αLacCer antibody (1:500; green) and rabbit <t>polyclonal</t> αNS4B antibody (1:150; red). Huh7.5 cells also were infected with HCV Jc1 (MOI of 1) and processed for confocal microscopy as described above. The boxed areas represent a magnified view for colocalization (yellow) of lactosylceramide and HCV NS4B protein. (B) Huh7.5 cells were infected with HCV Jc1 (MOI of 1) as described for panel A and processed for confocal microscopy 24 h, 48 h, and 72 h postinfection. For each infection time point, confocal images were taken of 20 representative cells. The intensity of lactosylceramide pixels was calculated with the JACoP plugin in ImageJ software. Each filled circle or square (24 h; mock or Jc1 infected), upper or lower triangle (48 h; mock or Jc1 infected), diamond (72 h; mock), or open circle (72 h; Jc1 infected) represents one cell.
    Rabbit Polyclonal Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 5570 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rabbit polyclonal antibodies
    Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using <t>polyclonal</t> rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 546, and counterstaining was performed with DAPI. Images are representative of three independent experiments.
    Rabbit Polyclonal Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2765 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher rabbit polyclonal antibody
    HKU1 neutralization by purified human IVIG and rabbit <t>polyclonal</t> antibodies to the HKU1 spike protein. HKU1 virus was incubated with the antibodies and then inoculated onto HTBE cells at 34 °C for 4 h. Following removal of the inoculum, the cells
    Rabbit Polyclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 2922 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone h3 antibody nuclear loading control and chip grade
    At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.
    Anti Histone H3 Antibody Nuclear Loading Control And Chip Grade, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 937 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti histone h3
    EP300 - and CREBBP - ZNF384 fusions resulted in loss of HAT activity, global <t>histone</t> acetylation deregulation, and sensitivity to HDAC inhibition. ( A , B ) HAT enzymatic activity was measured for various EP300 and CREBBP proteins: wild-type, truncated, and
    Anti Histone H3, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 3958 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher rabbit polyclonal antibodies
    Subcellular localization of WT/APP and 3M/APP by immunofluorescence microscopy. HCN-1A cells (A–L) and COS cells (M–R) were transfected with WT/APP (A–F) or 3M/APP (G–L). The inset in F is a twofold enlargement of a region showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na + / K + ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit <t>polyclonal</t> antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.
    Rabbit Polyclonal Antibodies, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1454 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti gapdh antibody loading control
    Interaction of tau with MSI proteins and <t>MSI1</t> silencing effect on tau levels. (a) Western blot of tau13 in MSI1 IP cytoplasm and nuclear fractions from P301L tau iHEK. (b) Western blot of tau13 in MSI2 IP cytoplasm and nuclear fractions from P301L tau iHEK. (c) Western blot of MSI1 from total lysate of P301L tau iHEK and relative quantification. (d) Western blot of MSI1 from total lysate of WT tau iHEK and relative quantification. (e) Western blot of MSI1, MSI2, and Tau13 in untreated and silenced WT tau iHEK total lysates. (f) MSI1 quantification. (g) MSI2 quantification. (h–i) HMW (75–250 kDa) tau and monomeric form (black arrow) quantification. Bar Graphs are used to show quantification of relative density as function of <t>GAPDH.</t> Student's t test has been use to determine statistical significance. Error bars represent SD
    Anti Gapdh Antibody Loading Control, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 796 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti flag antibody
    Ectopic expression of the assigned MmuPV1 ORFs in HEK293 cells. (A) MmuPV1 ORFs and their encoded protein size. (B) Protein sequences of E1^M1 and E1^M2. The N-terminal aa sequences of both E1^M1 and E1^M2 (grey box) are the same as the N-terminal E1 protein, but the N-terminal E1 in the E1^M1 is 23 aa residues longer than E1^M2 (grey box). The C-terminal aa residues of both E1^M1 and E1^M2 are the same and encoded by the same exon 2 being off-frame from E1 and thus totally differ from E1. (C) Expression of individual ORFs in HEK293 cells. Individual ORFs assigned were separately inserted into a mammalian expression vector in frame with a C-terminal <t>FLAG-tag.</t> Expression of the corresponding protein in HEK293 cells was examined by Western blotting using an <t>anti-FLAG</t> antibody at 24 h after transfection. (D) Expressed viral E6 and E7 are labile proteins in HEK293 cells. Cell lysates were prepared from HEK293 cells transfected by an E6 or E7 expression vector in the presence or absence of proteasome inhibitor MG132 for 6 h and examined by Western blotting as in (C). (E) FA staining of E6, E7, E2, E8^E2, E1^E4, E1^M1 and E1^M2 in HeLa cells by anti-FLAG M2 monoclonal antibody in combination with Alexa Flour488-labeled anti-mouse secondary antibody. The cell nuclei were counterstained by Hoechst 33342 dye.
    Anti Flag Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20915 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Agilent technologies rabbit polyclonal antibody
    Western blot of S100A6, using the <t>polyclonal</t> anti-S100A6 antibody (Dako) on lysates from prostate cancer cell lines. Lane 1. Du145; lane 2, LNCaP; lane 3, PC3. Note the protein expression in Du145 and PC3 cells, seen as an ∼10 kDa band, but its absence in the LNCaP cell line.
    Rabbit Polyclonal Antibody, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 92/100, based on 1579 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti cd31 antibody
    Depletion of Angpt2 from adipocytes alters subcutaneous fat distribution. a Diagram for generation of Angpt2 i∆Ad mice, inducible and specific deletion of Angpt2 in adipocytes by tamoxifen delivery into 4 week old mice and analyses in 8-week old mice. b Comparisons of Angpt2 mRNA expression in fractionized adipocytes (Ad) of SAT in WT and Angpt2 i∆Ad mice. c , d Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆Ad mice. n = 6 mice/group pooled from three independent experiments. e Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 ∆Ad mice. Four to six different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. f , g Representative images and comparisons of apoptosis (Caspase-3) and beiging (UCP1) in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 50 μm. h , i Representative images and comparison of indicated immune cell infiltration in SAT and VAT of WT and Angpt2 i∆Ad mice. Magnified view is shown in right panels. Scale bars, 30 μm. j , k Representative images and comparisons of vascular density and <t>CD31</t> + vessel area in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 100 μm. l Diagram for generation of Angpt2 i∆EC mice, inducible and specific deletion of Angpt2 in endothelial cells by tamoxifen delivery into 4 week old mice and analyses in 8 week old mice. m Comparisons of Angpt2 mRNA expression in stromal vascular fraction (SVF) of SAT in WT and Angpt2 i∆EC mice. n , o Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆EC mice. p Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 i∆EC mice. Four different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 3 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two-tailed Student’s t test. NS not significant.
    Anti Cd31 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa rabbit anti dsred
    Sexually dimorphic Mip neurons in the AG that regulate female receptivity. a Mip neurons in the AG from a female (above left) and a male (above right) carrying Mip-GAL4 , Mip6.0-GAL80 and <t>UAS-mCD8-GFP</t> stained by anti-GFP (green) and anti-nc82 (magenta) antibodies. The lower panels indicate the numbers and relative locations of Mip neuron somas in female (left) and male (right) AGs. The Mip neurons in the AG are grouped into six subsets: medial anterior lateral ( mAL ), ventral anterior lateral ( vAL ), ventral anterior medial ( vAM ), small ventral posterior medial ( s-vPM ), large ventral posterior medial ( l-vPM ), and small medial posterior medial ( s-mPM ). Filled and open circles indicate neurons positive and negative for anti-Mip, repectively. Scale bars, 50 μm. b The numbers of GFP-positive Mip neurons in the AGs of females (green) and males (red) carrying Mip-GAL4 , Mip6.0-GAL80 and UAS-mCD8-EGFP . Note the clear sexual dimorphism in the vAL and vAM neurons. c Negative images of TRIC labelling (anti-GFP) in the AGs of virgin (upper panels) and mated females (lower panels), indicating intracellular Ca 2+ transients. Scale bars, 10 μm. d The GFP intensities from vAM , l-vPM and SAG neurons of TRIC females show Ca 2+ activity in virgin (green) and mated females (red). e The frequencies of labelled (and therefore activated) neurons in the indicated Mip neuron subset of the AG from non-re-mating (green) and re-mating mosaic females (red). We used females carrying hsFLP , Mip-GAL4 , UAS-TrpA1 , <t>UAS-DsRed</t> and Tub FRT GAL80 FRT for stochastic manipulation (for details, see the text). NS indicates non-significance ( P > 0.05); ** P
    Rabbit Anti Dsred, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2070 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    Endogenous TRAPPC8 localizes to the centrosome/basal body. RPE cells were serum-starved for 24 h, fixed with methanol (upper two panels) or PFA (lower panel) and stained with rabbit polyclonal antibody against TRAPPC8 (green) and mouse monoclonal antibody against p150 Glued , rat monoclonal antibody against EB3 or mouse monoclonal antibody against acetylated tubulin (Ac tub), as indicated (red). DNA was stained with DAPI. Arrowheads and asterisks point to the centrosomes/basal bodies. Closed arrow indicates a primary cilium.

    Journal: Cilia

    Article Title: Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8

    doi: 10.1186/2046-2530-3-6

    Figure Lengend Snippet: Endogenous TRAPPC8 localizes to the centrosome/basal body. RPE cells were serum-starved for 24 h, fixed with methanol (upper two panels) or PFA (lower panel) and stained with rabbit polyclonal antibody against TRAPPC8 (green) and mouse monoclonal antibody against p150 Glued , rat monoclonal antibody against EB3 or mouse monoclonal antibody against acetylated tubulin (Ac tub), as indicated (red). DNA was stained with DAPI. Arrowheads and asterisks point to the centrosomes/basal bodies. Closed arrow indicates a primary cilium.

    Article Snippet: Cells were fixed with methanol and subjected to immunofluorescence microscopy as described [ ] using rabbit polyclonal antibody specific for TRAPPC8 (1:100 dilution; Sigma), rat monoclonal antibody specific for EB3 (1:300 dilution; Absea clone KT36), and mouse monoclonal antibodies specific for acetylated-tubulin (1:5,000 dilution; Sigma) and p150Glued (1:500 dilution; BD Biosciences).

    Techniques: Staining

    Western blot analysis and esiRNA-mediated depletion of TRAPPC8. (A) Western blot analysis of whole cell lysate from RPE cells, probed with rabbit polyclonal TRAPPC8 antibody. Molecular mass markers are shown in kDa to the left. (B) Western blot analysis of lysates from RPE cells treated with TRAPPC8-specific esiRNA or mock-transfected control cells. Blots were probed with antibodies specific for TRAPPC8 or α-tubulin (loading control). (C) Quantification of cilia in RPE cells depleted for TRAPPC8 using TRAPPC8-specific esiRNA. The cells were fixed with PFA and stained with acetylated tubulin antibody for visualization of cilia. Three independent experiments were conducted with 100 cells counted per condition per experiment. P value (*) = 0.0227 using unpaired t test. (D) Selected immunofluorescence micrographs of GFP-Rabin8 expressing mock-transfected control cells or cells depleted for TRAPPC8. Cells were first treated with mock or TRAPPC8-specific esiRNA and then transfected with GFP-Rabin8 plasmid. Following serum starvation for 1 h, cells were fixed with PFA and stained with antibody against p150 Glued to mark the centrosome (red). In Mock-transfected control cells, 92% of GFP-Rabin8 expressing cells showed GFP-Rabin8 at the centrosome whereas only 60% of the GFP-Rabin8 expressing TRAPPC8-depleted cells showed centrosomal GFP-Rabin8 localization (50 cells analyzed per condition).

    Journal: Cilia

    Article Title: Identification of conserved, centrosome-targeting ASH domains in TRAPPII complex subunits and TRAPPC8

    doi: 10.1186/2046-2530-3-6

    Figure Lengend Snippet: Western blot analysis and esiRNA-mediated depletion of TRAPPC8. (A) Western blot analysis of whole cell lysate from RPE cells, probed with rabbit polyclonal TRAPPC8 antibody. Molecular mass markers are shown in kDa to the left. (B) Western blot analysis of lysates from RPE cells treated with TRAPPC8-specific esiRNA or mock-transfected control cells. Blots were probed with antibodies specific for TRAPPC8 or α-tubulin (loading control). (C) Quantification of cilia in RPE cells depleted for TRAPPC8 using TRAPPC8-specific esiRNA. The cells were fixed with PFA and stained with acetylated tubulin antibody for visualization of cilia. Three independent experiments were conducted with 100 cells counted per condition per experiment. P value (*) = 0.0227 using unpaired t test. (D) Selected immunofluorescence micrographs of GFP-Rabin8 expressing mock-transfected control cells or cells depleted for TRAPPC8. Cells were first treated with mock or TRAPPC8-specific esiRNA and then transfected with GFP-Rabin8 plasmid. Following serum starvation for 1 h, cells were fixed with PFA and stained with antibody against p150 Glued to mark the centrosome (red). In Mock-transfected control cells, 92% of GFP-Rabin8 expressing cells showed GFP-Rabin8 at the centrosome whereas only 60% of the GFP-Rabin8 expressing TRAPPC8-depleted cells showed centrosomal GFP-Rabin8 localization (50 cells analyzed per condition).

    Article Snippet: Cells were fixed with methanol and subjected to immunofluorescence microscopy as described [ ] using rabbit polyclonal antibody specific for TRAPPC8 (1:100 dilution; Sigma), rat monoclonal antibody specific for EB3 (1:300 dilution; Absea clone KT36), and mouse monoclonal antibodies specific for acetylated-tubulin (1:5,000 dilution; Sigma) and p150Glued (1:500 dilution; BD Biosciences).

    Techniques: Western Blot, esiRNA, Transfection, Staining, Immunofluorescence, Expressing, Plasmid Preparation

    Western blot analysis of the laminin 5–type VII collagen–NC-1 complex immunoaffinity purified from collagenase extracts of amniotic membranes. Type VII collagen–NC-1 was first affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes. Eluted fractions of NC-1 were pooled, dialyzed against PBS, and passed over a mAb 6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 ( pLm5 ; lane 3 ), and anti-NC-1 ( mAb NP-32 , lane 4 ). The bound material shows the pattern obtained for purified laminin 5 (lane 1 ), which does not contain NC-1 (lane 2 ) and for purified NC-1 (not shown, but identical to lane 6 ). The nonbound fraction contains NC-1 only (lane 6 ) but no laminin 5 (lane 2 ).

    Journal: The Journal of Cell Biology

    Article Title: Laminin 5 Binds the NC-1 Domain of Type VII Collagen

    doi:

    Figure Lengend Snippet: Western blot analysis of the laminin 5–type VII collagen–NC-1 complex immunoaffinity purified from collagenase extracts of amniotic membranes. Type VII collagen–NC-1 was first affinity purified on mAb NP-32 from collagenase extracts of amniotic membranes. Eluted fractions of NC-1 were pooled, dialyzed against PBS, and passed over a mAb 6F12 affinity chromatography column. Bound materials were immunoblotted with polyclonal anti-laminin 5 ( pLm5 ; lane 3 ), and anti-NC-1 ( mAb NP-32 , lane 4 ). The bound material shows the pattern obtained for purified laminin 5 (lane 1 ), which does not contain NC-1 (lane 2 ) and for purified NC-1 (not shown, but identical to lane 6 ). The nonbound fraction contains NC-1 only (lane 6 ) but no laminin 5 (lane 2 ).

    Article Snippet: Antibodies Preparation and characterization of laminin 5 and 6 antibodies mAb BM165 (anti-α3 chain), mAb SF12 (anti-γ2), mAb 545 (anti-β1), and polyclonal antibody (pAb)1 anti-laminin 5 have been described elsewhere ( ; ). mAb NP32 to the NC-I domain of type VIl procollagen, and pAb made to the whole type VII procollagen molecule have been previously described ( ; ). pAb anti-laminin 1 was purchased from Sigma Chimie (St. Quentin Fallavier, France).

    Techniques: Western Blot, Purification, Affinity Purification, Affinity Column

    Western blot analysis of the complex of laminin 5 and NC-1 produced in solution. Laminin 5 alone (lanes A1 and B1 ), NC-1 alone (lanes A2 and B2 ), a mixture of laminin 5 and NC-1 (lanes A3 and B3 ), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4 ) were immunoprecipitated with anti-α3 mAb (BM-165). The contents of the precipitates were separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using ( A ) polyclonal anti-laminin 5 or ( B ) monoclonal anti-NC-1 (mAb NP-32).

    Journal: The Journal of Cell Biology

    Article Title: Laminin 5 Binds the NC-1 Domain of Type VII Collagen

    doi:

    Figure Lengend Snippet: Western blot analysis of the complex of laminin 5 and NC-1 produced in solution. Laminin 5 alone (lanes A1 and B1 ), NC-1 alone (lanes A2 and B2 ), a mixture of laminin 5 and NC-1 (lanes A3 and B3 ), or a mixture of laminin 5 and heat denatured NC-1 (lanes A4 and B4 ) were immunoprecipitated with anti-α3 mAb (BM-165). The contents of the precipitates were separated by SDS-PAGE after disulfide bond reduction and visualized by Western analysis using ( A ) polyclonal anti-laminin 5 or ( B ) monoclonal anti-NC-1 (mAb NP-32).

    Article Snippet: Antibodies Preparation and characterization of laminin 5 and 6 antibodies mAb BM165 (anti-α3 chain), mAb SF12 (anti-γ2), mAb 545 (anti-β1), and polyclonal antibody (pAb)1 anti-laminin 5 have been described elsewhere ( ; ). mAb NP32 to the NC-I domain of type VIl procollagen, and pAb made to the whole type VII procollagen molecule have been previously described ( ; ). pAb anti-laminin 1 was purchased from Sigma Chimie (St. Quentin Fallavier, France).

    Techniques: Western Blot, Produced, Immunoprecipitation, SDS Page

    In situ analysis of MeCP2 in cells and tissue. A ) Mouse myoblasts (C2C12 cells) were transiently transfected with GFP-MECP2 (human) and fixed using formaldehyde. MeCP2 was then detected with our monoclonal antibodies (undiluted) and our rabbit polyclonal antibody (1∶500). The first row shows the DNA counterstain (DAPI) of transfected and untransfected cells (green). The row underneath shows the signal obtained by our antibody staining (red). The third row shows the localization of the transfected GFP-MECP2 (blue). The merge contains an overlay of the antibody staining, the fluorescent signal of GFP-MECP2 and the DNA counterstain. Scale bar 20 µm. B ) Mouse wild type brain sections (25 µm) were stained using our antibodies. The first row shows the DNA counterstain with DAPI highlighting heterochromatic regions. The central row shows the signal obtained by immunofluorescence with our antibodies. The last row shows an overlay of DAPI and MeCP2. Scale bar 20 µm. C ) Mouse MeCP2 hemizygous null brain sections (25 µm) were stained as described above as a negative control. Mouse anti B23 antibody was used as a positive control (consecutive section when testing mouse monoclonal anti MeCP2). Scale bar 40 µm.

    Journal: PLoS ONE

    Article Title: Generation and Characterization of Rat and Mouse Monoclonal Antibodies Specific for MeCP2 and Their Use in X-Inactivation Studies

    doi: 10.1371/journal.pone.0026499

    Figure Lengend Snippet: In situ analysis of MeCP2 in cells and tissue. A ) Mouse myoblasts (C2C12 cells) were transiently transfected with GFP-MECP2 (human) and fixed using formaldehyde. MeCP2 was then detected with our monoclonal antibodies (undiluted) and our rabbit polyclonal antibody (1∶500). The first row shows the DNA counterstain (DAPI) of transfected and untransfected cells (green). The row underneath shows the signal obtained by our antibody staining (red). The third row shows the localization of the transfected GFP-MECP2 (blue). The merge contains an overlay of the antibody staining, the fluorescent signal of GFP-MECP2 and the DNA counterstain. Scale bar 20 µm. B ) Mouse wild type brain sections (25 µm) were stained using our antibodies. The first row shows the DNA counterstain with DAPI highlighting heterochromatic regions. The central row shows the signal obtained by immunofluorescence with our antibodies. The last row shows an overlay of DAPI and MeCP2. Scale bar 20 µm. C ) Mouse MeCP2 hemizygous null brain sections (25 µm) were stained as described above as a negative control. Mouse anti B23 antibody was used as a positive control (consecutive section when testing mouse monoclonal anti MeCP2). Scale bar 40 µm.

    Article Snippet: The slides were equilibrated in PBS after heating and incubated with the following antibodies: anti MeCP2 mouse monoclonal (undiluted), rat monoclonal (undiluted), rabbit polyclonal (1∶500), anti B23 mouse monoclonal (Sigma, St. Louis, MO, USA, 1∶1,000) and anti tyrosine hydroxylase rabbit antibody (AB152, Millipore, Billerica, MA, USA) Both, primary and secondary antibodies were complemented with 0.1% Triton X-100 and 1% BSA.

    Techniques: In Situ, Transfection, Staining, Immunofluorescence, Negative Control, Positive Control

    The effect of ATRA treatment on expression of ANXA2 and S100A10 mRNA and protein levels. The effect on ANXA2 and S100A10 expression following 1–6 days ATRA treatment (1, 5 μM) in OAW28 ( a and c ) and OV-90 ( b and d ) cells. ANXA2 and S100A10 expression was assessed using 2 -∆∆CT quantitation method and normalised to housekeeping gene β-actin using no treatment control as a calibrator. Data is from 2 to 4 independent experiments ( n = 4–12). Statistical significance was determined using one-way ANOVA and the Tukey multiple comparison post hoc test. Western blots for annexin A2 and S100A10 ( e and f ) following 6 days ATRA treatment (1, 5 μM). Annexin A2 bands at 38 kDa, S100A10 at 11 kDa and β-actin bands at 42 kDa are shown for OAW28 ( e ) and OV-90 ( f ) cells. Equal amounts protein (5 μg for annexin A2 15 μg S100A10 were run on a 4–12% SDS-PAGE gel and immunoblotted with mouse monoclonal antibodies to annexin A2 (1/2000, BD Biosciences) or S100A10 (1/2000, BD Biosciences). A rabbit polyclonal antibody was used detect β-actin (1/2000, ab8227, Abcam). Numbers below western bands in e and f are fold changes relative to the control treatment. Westerns blots are representative of two independent experiments

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: Anti-tumour effects of all-trans retinoid acid on serous ovarian cancer

    doi: 10.1186/s13046-018-1017-7

    Figure Lengend Snippet: The effect of ATRA treatment on expression of ANXA2 and S100A10 mRNA and protein levels. The effect on ANXA2 and S100A10 expression following 1–6 days ATRA treatment (1, 5 μM) in OAW28 ( a and c ) and OV-90 ( b and d ) cells. ANXA2 and S100A10 expression was assessed using 2 -∆∆CT quantitation method and normalised to housekeeping gene β-actin using no treatment control as a calibrator. Data is from 2 to 4 independent experiments ( n = 4–12). Statistical significance was determined using one-way ANOVA and the Tukey multiple comparison post hoc test. Western blots for annexin A2 and S100A10 ( e and f ) following 6 days ATRA treatment (1, 5 μM). Annexin A2 bands at 38 kDa, S100A10 at 11 kDa and β-actin bands at 42 kDa are shown for OAW28 ( e ) and OV-90 ( f ) cells. Equal amounts protein (5 μg for annexin A2 15 μg S100A10 were run on a 4–12% SDS-PAGE gel and immunoblotted with mouse monoclonal antibodies to annexin A2 (1/2000, BD Biosciences) or S100A10 (1/2000, BD Biosciences). A rabbit polyclonal antibody was used detect β-actin (1/2000, ab8227, Abcam). Numbers below western bands in e and f are fold changes relative to the control treatment. Westerns blots are representative of two independent experiments

    Article Snippet: Proteins bands were detected with mouse monoclonal antibodies to annexin A2 (1/2000, Clone 5, 610069, BD Biosciences, North Ryde, NSW, Australia) or S100A10 (1/2000, Clone 148, 610070, BD Biosciences), anti-mouse IgG peroxidase-conjugated secondary antibody (1/4000, A0168, Sigma Aldrich), enhanced chemiluminescence (GE Healthcare), and ChemiDoc™ MP Imaging System with ImageLab™ software (Bio-Rad, Hercules, CA, USA) [ ]. β-actin, used as a loading control was detected using a rabbit polyclonal antibody to β-actin (1/2000, ab8227, Abcam, Cambridge, MA, USA) and anti-rabbit IgG peroxidase-conjugated secondary antibody (1/4000, AP132P, Merck, Millipore, Bayswater, VIC, Australia).

    Techniques: Expressing, Quantitation Assay, Western Blot, SDS Page

    BOK is undetectable in WEHI7 and p53 −/− lymphoma cells. Whole-cell lysis of Bax −/− Bak1 −/− and Bax −/− Bak1 −/− WEH7 cells (upper panel) and p53 −/− T lymphoma cells (lower panel), were treated with 1 µM Dex for the indicated times and analyzed by western blot. Membranes were incubated with a rabbit anti–BOK polyclonal antibody and subsequently with a monoclonal antibody to ACTIN. Lysates from Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bok −/− mouse embryonic fibroblast lines (MEFs) were used as positive and negative controls. The results of one of two independent experiments are shown.

    Journal: Cell Death & Disease

    Article Title: Glucocorticoids can induce BIM to trigger apoptosis in the absence of BAX and BAK1

    doi: 10.1038/s41419-020-2599-5

    Figure Lengend Snippet: BOK is undetectable in WEHI7 and p53 −/− lymphoma cells. Whole-cell lysis of Bax −/− Bak1 −/− and Bax −/− Bak1 −/− WEH7 cells (upper panel) and p53 −/− T lymphoma cells (lower panel), were treated with 1 µM Dex for the indicated times and analyzed by western blot. Membranes were incubated with a rabbit anti–BOK polyclonal antibody and subsequently with a monoclonal antibody to ACTIN. Lysates from Bax −/− Bak1 −/− and Bax −/− Bak1 −/− Bok −/− mouse embryonic fibroblast lines (MEFs) were used as positive and negative controls. The results of one of two independent experiments are shown.

    Article Snippet: Ryan, Monash University), Cleaved CASP3 (#9661, Cell Signaling Technology), Cleaved CASP-9 (#9509, Cell Signaling Technology), PUMA (#ab9645, Abcam), VDAC1(Abcam, ab15895), Rabbit polyclonal antibodies raised against amino acids 19–32 of mBOK (gift from Francine Ke, WEHI ), BID (BD Biosciences, #559681), BCLXL (#2764, Cell Signaling Technology), BIM, APAF1, BMF, and CASP9 are from in house (L. O’Reilly, WEHI).

    Techniques: Lysis, Western Blot, Incubation

    Histological characterization of LRRK2 KO kidneys. ( A ) Confocal imaging of WT, LRRK2-KO (5- to 6-month-old, n = 3) for cathepsin D (rabbit polyclonal antibody, AlexaFluor 568 secondary antibody), LAMP1 (rat monoclonal antibody, AlexaFluor 488 secondary antibody) and DAPI staining. Scale bar: 20 μm. ( B ) Representative Airy scan images of the same WT and LRRK2-KO kidneys for cathepsin D, LAMP1 and DAPI staining. Scale bar: 10 μm. ( C ) Confocal imaging of the same WT and LRRK2-KO kidneys for cathepsin D (goat polyclonal antibody, AlexaFluor 568 secondary antibody) and M6PR (rabbit monoclonal antibody, AlexaFluor 488 secondary antibody) and DAPI staining. Scale bar: 20 μm. ( D ) Quantification of cathepsin D and LAMP1 colocalization in the kidney sections shown in panel (A). ( E ) Quantification of cathepsin D and M6PR colocalization in the kidney sections shown in ( C ). Data points represent Pearson’s correlation coefficient from at least five images per animal, with multiple cells per image, n = 3 mice per genotype. ( F ) Number of cathepsin D punctae per nuclei and area (μm 2 ) of cathepsin D ( G ) and LAMP1 ( H ) positive vesicles. ( I ) Immunoblot analysis of WT and LRRK2-KO kidney lysates (12-month-old total homogenates, n = 5) using CI-M6PR and LAMP1 antibodies. Quantifications of CI-M6PR ( J ) and of LAMP1 ( K ) immunoblots normalized for the loading control β-actin.

    Journal: Human Molecular Genetics

    Article Title: Proteomic analysis reveals co-ordinated alterations in protein synthesis and degradation pathways in LRRK2 knockout mice

    doi: 10.1093/hmg/ddy232

    Figure Lengend Snippet: Histological characterization of LRRK2 KO kidneys. ( A ) Confocal imaging of WT, LRRK2-KO (5- to 6-month-old, n = 3) for cathepsin D (rabbit polyclonal antibody, AlexaFluor 568 secondary antibody), LAMP1 (rat monoclonal antibody, AlexaFluor 488 secondary antibody) and DAPI staining. Scale bar: 20 μm. ( B ) Representative Airy scan images of the same WT and LRRK2-KO kidneys for cathepsin D, LAMP1 and DAPI staining. Scale bar: 10 μm. ( C ) Confocal imaging of the same WT and LRRK2-KO kidneys for cathepsin D (goat polyclonal antibody, AlexaFluor 568 secondary antibody) and M6PR (rabbit monoclonal antibody, AlexaFluor 488 secondary antibody) and DAPI staining. Scale bar: 20 μm. ( D ) Quantification of cathepsin D and LAMP1 colocalization in the kidney sections shown in panel (A). ( E ) Quantification of cathepsin D and M6PR colocalization in the kidney sections shown in ( C ). Data points represent Pearson’s correlation coefficient from at least five images per animal, with multiple cells per image, n = 3 mice per genotype. ( F ) Number of cathepsin D punctae per nuclei and area (μm 2 ) of cathepsin D ( G ) and LAMP1 ( H ) positive vesicles. ( I ) Immunoblot analysis of WT and LRRK2-KO kidney lysates (12-month-old total homogenates, n = 5) using CI-M6PR and LAMP1 antibodies. Quantifications of CI-M6PR ( J ) and of LAMP1 ( K ) immunoblots normalized for the loading control β-actin.

    Article Snippet: Antibodies The following antibodies used for immunoblot were diluted in a 1:1 mix of Odyssey Blocking Buffer (LI-COR) and TBS containing 0.1% (v/v) Tween 20:rabbit polyclonal antibody to cathepsin D (Millipore, 219361, 1:1000), goat polyclonal antibody to cathepsin D C-20 (Santa Cruz, sc-6486, 1:200), rabbit polyclonal antibody to legumain (Abcam, ab125286, 1:1000), mouse monoclonal antibody to gephyrin (Synaptic System, 147 111, 1:2000), mouse monoclonal antibody to β-actin (Sigma, A5316, 1:10 000), rabbit polyclonal antibody to α/β-tubulin (Cell Signaling, 2148, 1:1000), mouse monoclonal antibody to acetylated-tubulin (Sigma, T7451, 1:1000), rabbit polyclonal antibody to coronin 1C (Proteintech, 14749-1-AP, 1:1000), mouse monoclonal antibody to NAA15 (LSBio, LS-C342562, 1:1000), rabbit polyclonal to EEF1G (Abcam, ab72368, 1:1000), mouse monoclonal to HNRNPK antibody (Abcam, ab23644, 1:1000) and rabbit polyclonal to LC3B (Abcam, ab51520, 1:2000).

    Techniques: Imaging, Staining, Mouse Assay, Western Blot

    The requirements for the translocation of ICP0 from the nucleus to the cytoplasm. (A) ICP0 carrying the D199A substitution encoded by R7914 is not translocated to the cytoplasm late in infection of HEL fibroblasts. Replicate slide cultures of HEL fibroblasts were infected with 20 PFU of HSV-1(F), the recombinant virus R7914, or the repaired virus R7915 per cell and maintained at 37°C. At 12 h after infection, the cells were fixed and reacted first with polyclonal rabbit serum directed against exon II of ICP0 and second with FITC-conjugated goat anti-rabbit immunoglobulin antibodies. Sequential fields were examined in a Zeiss confocal microscope, and the numbers of cells exhibiting nuclear, cytoplasmic, or both nuclear and cytoplasmic localization of ICP0 were tabulated as shown in the histogram. The numbers above the bars indicate the numbers of cells showing a specific distribution of ICP0. (B) A viral gene function mediates the translocation of ICP0 from the nucleus to the cytoplasm during productive infection. Replicate slide cultures of HEL fibroblasts were exposed first to recombinant baculoviruses encoding wild-type or mutant ICP0 (D199A). After 2 h, cells were treated with 5 mM Na-butyrate. At 3 h after exposure to baculoviruses, the cells were exposed to the recombinant virus R7910, from which both copies of the α0 gene had been deleted. After 15 h of exposure to baculoviruses alone or 12 h after infection with R7910, the cells were fixed and reacted with rabbit polyclonal antibody against ICP0 and mouse monoclonal antibody against gD and then reacted with FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG antibodies. Cell counts were done as described above, except that only cells exhibiting gD encoded by R7910 and ICP0 encoded by baculoviruses were counted.

    Journal: Journal of Virology

    Article Title: Requirements for the Nuclear-Cytoplasmic Translocation of Infected-Cell Protein 0 of Herpes Simplex Virus 1

    doi: 10.1128/JVI.75.8.3832-3840.2001

    Figure Lengend Snippet: The requirements for the translocation of ICP0 from the nucleus to the cytoplasm. (A) ICP0 carrying the D199A substitution encoded by R7914 is not translocated to the cytoplasm late in infection of HEL fibroblasts. Replicate slide cultures of HEL fibroblasts were infected with 20 PFU of HSV-1(F), the recombinant virus R7914, or the repaired virus R7915 per cell and maintained at 37°C. At 12 h after infection, the cells were fixed and reacted first with polyclonal rabbit serum directed against exon II of ICP0 and second with FITC-conjugated goat anti-rabbit immunoglobulin antibodies. Sequential fields were examined in a Zeiss confocal microscope, and the numbers of cells exhibiting nuclear, cytoplasmic, or both nuclear and cytoplasmic localization of ICP0 were tabulated as shown in the histogram. The numbers above the bars indicate the numbers of cells showing a specific distribution of ICP0. (B) A viral gene function mediates the translocation of ICP0 from the nucleus to the cytoplasm during productive infection. Replicate slide cultures of HEL fibroblasts were exposed first to recombinant baculoviruses encoding wild-type or mutant ICP0 (D199A). After 2 h, cells were treated with 5 mM Na-butyrate. At 3 h after exposure to baculoviruses, the cells were exposed to the recombinant virus R7910, from which both copies of the α0 gene had been deleted. After 15 h of exposure to baculoviruses alone or 12 h after infection with R7910, the cells were fixed and reacted with rabbit polyclonal antibody against ICP0 and mouse monoclonal antibody against gD and then reacted with FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG antibodies. Cell counts were done as described above, except that only cells exhibiting gD encoded by R7910 and ICP0 encoded by baculoviruses were counted.

    Article Snippet: The rabbit polyclonal antibody to ICP0 , the mouse monoclonal antibody to PML (Santa Cruz Biotechnology, Santa Cruz, Calif.), and the rabbit polyclonal antibody to proteasomal proteins (Affiniti Research Products Ltd., Mamhead, Exeter, Devon, United Kingdom; catalogue no. PW 8155) were used at 1:1,000, 1:200, and 1:2,500 dilutions, respectively.

    Techniques: Translocation Assay, Infection, Recombinant, Microscopy, Mutagenesis

    Peli1 interacts specifically with Mdmx and promotes Mdmx ubiquitination (A) Peli1 cannot bind to p53. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or together with FH-Mdmx or FLAG-p53 were subjected to western blot with anti-Myc (top) and anti-FLAG antibody (lower). (B) Peli1 cannot bind to Mdm2. Whole cell extracts or immunoprecipitates with anti-Myc antibody from H1299 cells transfected with FH-Peli1 alone or together with Myc-Mdmx or Myc-Mdm2 were subjected to western blot with anti-Myc (top) and anti-FLAG antibody (lower). (C) Peli1 cannot promote Mdmx degradation. Western blot analysis of cell extracts from the H1299 cells transfected with FH-Mdmx alone or together with increased Myc-Peli1 with anti-HA (top) and anti-Myc antibody (middle). GFP was used as loading control. (D) Peli1 promotes Mdmx poly-ubiquitination. FH-Mdmx was transfected alone (lane 1), or with HA-His-Ubiquitin (HH-Ub, lane 2), or with Myc-Peli1 (lane 3), or with Myc-Peli1 and HH-Ub (lane 4), or with Myc-Peli1 mutant (1–310aa) and HH-Ub (lane 5) in H1299 cells. The cell lysates were immunoprecipitated with Ni-NTA agarose followed by western blot with anti-Mdmx polyclonal antibodies (upper panel). The crude cell extracts were also detected with anti-HA (lower panel, top) and anti-Myc (lower panel, middle) monoclonal antibodies. GFP was used as loading control. (E) Peli1 promotes Mdmx mono-ubiquitination. FH-Mdmx was transfected alone (lane 4), or with HH-Ub (lane 1), or with Myc-Peli1 and HH-Ub (lane 2), or with Myc-Peli1 mutant (1–310aa) and HH-Ub (lane 3) in H1299 cells. The crude cell extracts and immunoprecipitates from FLAG/M2 beads were subjected to western blot with anti-Mdmx polyclonal antibody (upper panel), anti-HA (lower panel, top) and anti-Myc (lower panel, middle) monoclonal antibodies. GFP was used as loading control.

    Journal: Cancer research

    Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

    doi: 10.1158/0008-5472.CAN-17-3531

    Figure Lengend Snippet: Peli1 interacts specifically with Mdmx and promotes Mdmx ubiquitination (A) Peli1 cannot bind to p53. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or together with FH-Mdmx or FLAG-p53 were subjected to western blot with anti-Myc (top) and anti-FLAG antibody (lower). (B) Peli1 cannot bind to Mdm2. Whole cell extracts or immunoprecipitates with anti-Myc antibody from H1299 cells transfected with FH-Peli1 alone or together with Myc-Mdmx or Myc-Mdm2 were subjected to western blot with anti-Myc (top) and anti-FLAG antibody (lower). (C) Peli1 cannot promote Mdmx degradation. Western blot analysis of cell extracts from the H1299 cells transfected with FH-Mdmx alone or together with increased Myc-Peli1 with anti-HA (top) and anti-Myc antibody (middle). GFP was used as loading control. (D) Peli1 promotes Mdmx poly-ubiquitination. FH-Mdmx was transfected alone (lane 1), or with HA-His-Ubiquitin (HH-Ub, lane 2), or with Myc-Peli1 (lane 3), or with Myc-Peli1 and HH-Ub (lane 4), or with Myc-Peli1 mutant (1–310aa) and HH-Ub (lane 5) in H1299 cells. The cell lysates were immunoprecipitated with Ni-NTA agarose followed by western blot with anti-Mdmx polyclonal antibodies (upper panel). The crude cell extracts were also detected with anti-HA (lower panel, top) and anti-Myc (lower panel, middle) monoclonal antibodies. GFP was used as loading control. (E) Peli1 promotes Mdmx mono-ubiquitination. FH-Mdmx was transfected alone (lane 4), or with HH-Ub (lane 1), or with Myc-Peli1 and HH-Ub (lane 2), or with Myc-Peli1 mutant (1–310aa) and HH-Ub (lane 3) in H1299 cells. The crude cell extracts and immunoprecipitates from FLAG/M2 beads were subjected to western blot with anti-Mdmx polyclonal antibody (upper panel), anti-HA (lower panel, top) and anti-Myc (lower panel, middle) monoclonal antibodies. GFP was used as loading control.

    Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

    Techniques: Transfection, Western Blot, Mutagenesis, Immunoprecipitation

    Identification of Peli1 as a bona fide Mdmx interacting protein in cancer cells (A) Silver staining of affinity-purified Mdmx complexes from the cell extracts of the FH-Mdmx A375 stable cells and the parental A375 cells. Mdmx-interacting partners were identified by mass spectrometry, and the Peli1 peptide sequences are presented. (B) Co-immunoprecipitation of FH-Peli1 with Myc-Mdmx from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Mdmx alone or with Myc-Mdmx and FH-Peli1 together were subjected to western blot with anti-Myc (top) and anti-HA antibody (lower). (C) Co-immunoprecipitation of FH-Mdmx with Myc-Peli1 from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or with Myc-Peli1 and FH-Mdmx together were subjected to western blot with anti-HA (top) and anti-Myc antibody (lower). (D) Direct interactions of GST-Peli1 with Mdmx. The GST-Peli1 fusion protein and GST alone were used in a GST pull-down assay with the FH-Mdmx protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Peli1 fusion protein. (E) Direct interactions of GST-Mdmx with Peli1. The GST-Mdmx fusion protein and GST alone were used in a GST pull-down assay with the FH-Peli1 protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Mdmx fusion protein. (F) Co-immunoprecipitation of endogenous Mdmx with Peli1 from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) and immunoprecipitates with a control IgG (lane 2) or a Peli1-specific antibody (lane 3) by anti-Peli1 monoclonal antibody (top) or anti-Mdmx polyclonal antibody (lower). (G) Co-immunoprecipitation of endogenous Peli1 with Mdmx from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) or immunoprecipitates with control IgG (lane 2) or anti-Mdmx polyclonal antibody (lane 3) by anti-Mdmx polyclonal antibody (top) or anti-Peli1 monoclonal antibody (lower).

    Journal: Cancer research

    Article Title: Peli1 modulates the subcellular localization and activity of Mdmx

    doi: 10.1158/0008-5472.CAN-17-3531

    Figure Lengend Snippet: Identification of Peli1 as a bona fide Mdmx interacting protein in cancer cells (A) Silver staining of affinity-purified Mdmx complexes from the cell extracts of the FH-Mdmx A375 stable cells and the parental A375 cells. Mdmx-interacting partners were identified by mass spectrometry, and the Peli1 peptide sequences are presented. (B) Co-immunoprecipitation of FH-Peli1 with Myc-Mdmx from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Mdmx alone or with Myc-Mdmx and FH-Peli1 together were subjected to western blot with anti-Myc (top) and anti-HA antibody (lower). (C) Co-immunoprecipitation of FH-Mdmx with Myc-Peli1 from H1299 cells. Whole cell extracts or immunoprecipitates with FLAG/M2 beads from H1299 cells transfected with Myc-Peli1 alone or with Myc-Peli1 and FH-Mdmx together were subjected to western blot with anti-HA (top) and anti-Myc antibody (lower). (D) Direct interactions of GST-Peli1 with Mdmx. The GST-Peli1 fusion protein and GST alone were used in a GST pull-down assay with the FH-Mdmx protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Peli1 fusion protein. (E) Direct interactions of GST-Mdmx with Peli1. The GST-Mdmx fusion protein and GST alone were used in a GST pull-down assay with the FH-Peli1 protein purified from H1299 cells. Elutes from GST beads were subjected to western blot with anti-HA antibody (top) after Ponceau S staining (lower). Asterisk indicates GST-Mdmx fusion protein. (F) Co-immunoprecipitation of endogenous Mdmx with Peli1 from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) and immunoprecipitates with a control IgG (lane 2) or a Peli1-specific antibody (lane 3) by anti-Peli1 monoclonal antibody (top) or anti-Mdmx polyclonal antibody (lower). (G) Co-immunoprecipitation of endogenous Peli1 with Mdmx from A375 melanoma cells. Western blot analysis of whole cell extract (lane 1) or immunoprecipitates with control IgG (lane 2) or anti-Mdmx polyclonal antibody (lane 3) by anti-Mdmx polyclonal antibody (top) or anti-Peli1 monoclonal antibody (lower).

    Article Snippet: Mouse monoclonal antibody for Peli1 (F-7), p53 (DO-1), Mdm2 (SMP14) and Myc (9E10) and rabbit polyclonal antibodies for p21 (C19) and Puma (H-136) were purchased from Santa Cruz Biotechnology.

    Techniques: Silver Staining, Affinity Purification, Mass Spectrometry, Immunoprecipitation, Transfection, Western Blot, Pull Down Assay, Purification, Staining

    Representative Western blots showing the time course of cyclin D1, E, and A protein expression induced by FCS (10%) in cultured rat aortic smooth muscle cells. Equal amounts of protein from nuclear extracts were subjected to SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with polyclonal antibodies against G1 phase cyclin D1 (top), and cyclin E (center) and S phase cyclin A (bottom). Similar results were obtained in two additional experiments.

    Journal: British Journal of Pharmacology

    Article Title: Growth-inhibitory effect of cyclic GMP- and cyclic AMP-dependent vasodilators on rat vascular smooth muscle cells: effect on cell cycle and cyclin expression

    doi: 10.1038/sj.bjp.0702305

    Figure Lengend Snippet: Representative Western blots showing the time course of cyclin D1, E, and A protein expression induced by FCS (10%) in cultured rat aortic smooth muscle cells. Equal amounts of protein from nuclear extracts were subjected to SDS–PAGE, transferred to nitrocellulose membranes, and immunoblotted with polyclonal antibodies against G1 phase cyclin D1 (top), and cyclin E (center) and S phase cyclin A (bottom). Similar results were obtained in two additional experiments.

    Article Snippet: The immobilized cyclin D1, E and A proteins were detected by subsequent incubation with polyclonal rabbit antibodies directed against cyclin D1, E and A, respectively, (dilution 1 : 1000, overnight at 4°C; Santa Cruz, Heidelberg, Germany) and then with a secondary polyclonal donkey anti-rabbit immunoglobulin antibody conjugated to horseradish peroxidase (dilution 1 : 10,000, 1 h at 22°C; Calbiochem).

    Techniques: Western Blot, Expressing, Cell Culture, SDS Page

    Lactosylceramide is associated with HCV NS4B protein. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αLacCer antibody (1:500; green) and rabbit polyclonal αNS4B antibody (1:150; red). Huh7.5 cells also were infected with HCV Jc1 (MOI of 1) and processed for confocal microscopy as described above. The boxed areas represent a magnified view for colocalization (yellow) of lactosylceramide and HCV NS4B protein. (B) Huh7.5 cells were infected with HCV Jc1 (MOI of 1) as described for panel A and processed for confocal microscopy 24 h, 48 h, and 72 h postinfection. For each infection time point, confocal images were taken of 20 representative cells. The intensity of lactosylceramide pixels was calculated with the JACoP plugin in ImageJ software. Each filled circle or square (24 h; mock or Jc1 infected), upper or lower triangle (48 h; mock or Jc1 infected), diamond (72 h; mock), or open circle (72 h; Jc1 infected) represents one cell.

    Journal: Journal of Virology

    Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

    doi: 10.1128/JVI.00970-14

    Figure Lengend Snippet: Lactosylceramide is associated with HCV NS4B protein. (A) Parental Huh7.5 and HCV Con1 (genotype 1b) replicon cells were grown for 48 h and processed for confocal microscopy with mouse monoclonal αLacCer antibody (1:500; green) and rabbit polyclonal αNS4B antibody (1:150; red). Huh7.5 cells also were infected with HCV Jc1 (MOI of 1) and processed for confocal microscopy as described above. The boxed areas represent a magnified view for colocalization (yellow) of lactosylceramide and HCV NS4B protein. (B) Huh7.5 cells were infected with HCV Jc1 (MOI of 1) as described for panel A and processed for confocal microscopy 24 h, 48 h, and 72 h postinfection. For each infection time point, confocal images were taken of 20 representative cells. The intensity of lactosylceramide pixels was calculated with the JACoP plugin in ImageJ software. Each filled circle or square (24 h; mock or Jc1 infected), upper or lower triangle (48 h; mock or Jc1 infected), diamond (72 h; mock), or open circle (72 h; Jc1 infected) represents one cell.

    Article Snippet: Rabbit polyclonal antibody to calnexin was purchased from Cell Signaling (Danvers, MA).

    Techniques: Confocal Microscopy, Infection, Software

    ). At 48 h postinfection, the cells were processed for confocal microscopy with mouse monoclonal αPI4P antibody (green). NS5A was detected via mCherry fluorescence. Alternatively, HCV Con1 replicon cells were grown for 48 h and stained with mouse monoclonal α-PI4P antibody (red) and rabbit polyclonal antibody against NS4B (green). The boxed areas are a magnified view for colocalization (yellow) of HCV NS5A or NS4B protein with PI4P. (C) Diagram of the de novo ) are two pharmacological inhibitors of UGCGC. FAPP2 is highlighted in gray and carries glucosylceramide from the cis -Golgi to the trans -Golgi network for conversion into lactosylceramide and other glycosphingolipids. CoA, coenzyme A.

    Journal: Journal of Virology

    Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

    doi: 10.1128/JVI.00970-14

    Figure Lengend Snippet: ). At 48 h postinfection, the cells were processed for confocal microscopy with mouse monoclonal αPI4P antibody (green). NS5A was detected via mCherry fluorescence. Alternatively, HCV Con1 replicon cells were grown for 48 h and stained with mouse monoclonal α-PI4P antibody (red) and rabbit polyclonal antibody against NS4B (green). The boxed areas are a magnified view for colocalization (yellow) of HCV NS5A or NS4B protein with PI4P. (C) Diagram of the de novo ) are two pharmacological inhibitors of UGCGC. FAPP2 is highlighted in gray and carries glucosylceramide from the cis -Golgi to the trans -Golgi network for conversion into lactosylceramide and other glycosphingolipids. CoA, coenzyme A.

    Article Snippet: Rabbit polyclonal antibody to calnexin was purchased from Cell Signaling (Danvers, MA).

    Techniques: Confocal Microscopy, Fluorescence, Staining

    ) virus-infected cells were grown as described for panel A and processed for confocal microscopy with mouse monoclonal antibody against dsRNA (1: 200; red) and rabbit polyclonal αFAPP2 antibody (1:100; green). The boxed areas indicate the magnified view for colocalization (yellow color) of HCV NS5A (A) or dsRNA (B) with FAPP2 protein. (C) FAPP2 cofractionates with HCV replicase NS5A protein in the detergent-resistant membrane fraction. Con1 replicon cell lysates were left untreated or were treated with 1% NP-40 on ice and subjected to membrane floatation. Proteins from pooled fractions (1 to 4 and 5 to 9) were separated by SDS-PAGE, followed by immunoblotting with antibodies against FAPP2, SPTLC1, NS5A, calnexin, or GAPDH. Numbers 1 to 4 refer to membrane (M) fractions, and numbers 5 to 8 refer to soluble (S) fractions.

    Journal: Journal of Virology

    Article Title: Modulation of Hepatitis C Virus Genome Replication by Glycosphingolipids and Four-Phosphate Adaptor Protein 2

    doi: 10.1128/JVI.00970-14

    Figure Lengend Snippet: ) virus-infected cells were grown as described for panel A and processed for confocal microscopy with mouse monoclonal antibody against dsRNA (1: 200; red) and rabbit polyclonal αFAPP2 antibody (1:100; green). The boxed areas indicate the magnified view for colocalization (yellow color) of HCV NS5A (A) or dsRNA (B) with FAPP2 protein. (C) FAPP2 cofractionates with HCV replicase NS5A protein in the detergent-resistant membrane fraction. Con1 replicon cell lysates were left untreated or were treated with 1% NP-40 on ice and subjected to membrane floatation. Proteins from pooled fractions (1 to 4 and 5 to 9) were separated by SDS-PAGE, followed by immunoblotting with antibodies against FAPP2, SPTLC1, NS5A, calnexin, or GAPDH. Numbers 1 to 4 refer to membrane (M) fractions, and numbers 5 to 8 refer to soluble (S) fractions.

    Article Snippet: Rabbit polyclonal antibody to calnexin was purchased from Cell Signaling (Danvers, MA).

    Techniques: Infection, Confocal Microscopy, SDS Page

    Fiber-specific changes in expression of activated AMPK and mitochondrial marker ATP synthase Since it was anticipated that exercise that was primarily endurance training would impact mitochondrial biogenesis, muscle AMPK activation and ATP synthase expression were quantified. Sections of muscle from each subject pre- and post-training were incubated with a monoclonal antibody against slow-twitch myosin heavy chain to identify the type 1 fibers on the section. A second rabbit polyclonal antibody against either phospho-AMPK or ATP synthase was added and incubated overnight at 4° C. Fluorescent tagged anti-rabbit IgG and anti-mouse IgG were added after the primary incubation and dual color images were obtained using a Leica confocal microscope. The signal intensity generated with either the phospho-AMPK or the ATP synthase antibody for the identified fiber type was quantified using digital imaging software (Quantity One from BioRad). At least thirty fibers had the signal intensity quantified for each antigen. Panel A displays the data for the control and metabolic syndrome subjects before and after training in the type 1 fibers. The expression of phospho-AMPK tended to be lower in the type 2x fibers for both controls and metabolic syndrome subjects, but ATP synthase expression appeared higher in 2x fibers. The activated AMPK was 2-fold increased after training for both groups in both fiber types. The ATP synthase was significantly increased by training in type 2x only in the metabolic syndrome group, indicating training-induced increased mitochondrial production, albeit not nearly to the level of increased activated AMPK. An asterix indicates significant post-training increase in expression compared to corresponding baseline (p

    Journal: Medicine and science in sports and exercise

    Article Title: Insulin Responsiveness in Metabolic Syndrome after Eight Weeks of Cycle Training

    doi: 10.1249/MSS.0b013e31829a6ce8

    Figure Lengend Snippet: Fiber-specific changes in expression of activated AMPK and mitochondrial marker ATP synthase Since it was anticipated that exercise that was primarily endurance training would impact mitochondrial biogenesis, muscle AMPK activation and ATP synthase expression were quantified. Sections of muscle from each subject pre- and post-training were incubated with a monoclonal antibody against slow-twitch myosin heavy chain to identify the type 1 fibers on the section. A second rabbit polyclonal antibody against either phospho-AMPK or ATP synthase was added and incubated overnight at 4° C. Fluorescent tagged anti-rabbit IgG and anti-mouse IgG were added after the primary incubation and dual color images were obtained using a Leica confocal microscope. The signal intensity generated with either the phospho-AMPK or the ATP synthase antibody for the identified fiber type was quantified using digital imaging software (Quantity One from BioRad). At least thirty fibers had the signal intensity quantified for each antigen. Panel A displays the data for the control and metabolic syndrome subjects before and after training in the type 1 fibers. The expression of phospho-AMPK tended to be lower in the type 2x fibers for both controls and metabolic syndrome subjects, but ATP synthase expression appeared higher in 2x fibers. The activated AMPK was 2-fold increased after training for both groups in both fiber types. The ATP synthase was significantly increased by training in type 2x only in the metabolic syndrome group, indicating training-induced increased mitochondrial production, albeit not nearly to the level of increased activated AMPK. An asterix indicates significant post-training increase in expression compared to corresponding baseline (p

    Article Snippet: Rabbit polyclonal antibodies specific for human IRS-1 phosphorylated at Tyr896 (#3070), Ser307 (#2491), Ser337 (#2580), Ser636 (#2388), and Ser1101 (#2385) were purchased from Cell Signaling.

    Techniques: Expressing, Marker, Activation Assay, Incubation, Microscopy, Generated, Imaging, Software

    Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using polyclonal rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 546, and counterstaining was performed with DAPI. Images are representative of three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: TRPV-5 Mediates a Receptor Activator of NF-?B (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption *

    doi: 10.1074/jbc.M109.075234

    Figure Lengend Snippet: Expression of Ca 2+ channels in human osteoclasts. At the end of the CBM cultures, immunofluorescence was performed on mature osteoclasts, using polyclonal rabbit antibodies directed against Ca 2+ channels (L-, T-, and R-channels and TRPV-5) and goat anti-rabbit secondary antibodies conjugated to Alexa Fluor 546, and counterstaining was performed with DAPI. Images are representative of three independent experiments.

    Article Snippet: Recombinant human macrophage-CSF and recombinant human granulocyte-macrophage-CSF were purchased from R & D Systems (Minneapolis, MN); goat polyclonal antibodies against calcitonin receptor (CTR) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mouse monoclonal antibodies against TRPV-5 were from Abcam (Cambridge, MA), and rabbit polyclonal antibodies were from Upstate Biotech Millipore (Billerica, MA); rabbit polyclonal antibodies against L-type and T-type Ca2+ channels were purchased from Sigma-Aldrich; those against R-type channels were from Novus Biologicals (Littleton, CO); and rabbit polyclonal antibodies against V-ATPase were purchased from Chemicon (Temecula, CA).

    Techniques: Expressing, Immunofluorescence

    Characterization of Loss-of-function INTS13 mutations in patient cells. a RT-qPCR shows that endogenous INTS13 transcript levels were significantly reduced relative to control cells in primary dermal fibroblasts of affected individual II.4 from Family 1. Data are mean +/-SD, n = 3. Statistical significance was calculated using a two-tailed unpaired t-test. b Primary dermal fibroblasts from individual II.4 from Family 1 were treated with 100 g/ml cycloheximide (CHX). Following treatment, RT-qPCR was done which shows that the INTS13 mRNA level was increased in treated cells. Data are mean +/-SEM, n = 3. Statistical significance was calculated using a one-tailed Student’s t-test. c Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 1 using two polyclonal antibodies designed against distinct regions of INTS13. No signal was detected in patient cells with the antibody directed against the C-terminus of INTS13 (C). A truncated protein was detected in patient cells by the antibody directed against the center of the protein (M). Actin was used as a loading control. d RT-qPCR shows that INTS13 transcript levels were significantly reduced in primary dermal fibroblast cells of affected individual II.4 from Family 2 relative to control cells. Data are mean +/-SD, n = 3. Statistical significance was calculated using a two-tailed unpaired t-test. e Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 2 shows reduced levels of INTS13 protein compared to control cells. f Western blot analysis of primary dermal fibroblasts from individual II.4 from Family 2 treated with MG132. Treated cells show a level of INTS13 protein comparable to control cells. * indicates a non-specific signal. g Multiciliated airway cells were obtained from nasal biopsies of three members of Family 1: the unaffected carrier mother (Con.) and the two affected children (II.4 and II.5). Probing for acetylated α-tubulin shows significantly shorter, less dense, and disorganized cilia in the affected individuals compared to the mother’s cells. h Bar graph representing the average length of nasal cilia. Data are mean +/-SEM, n = 200 cells for each individual. Statistical significance was calculated using a one-tailed t-test. *, **, ***, **** correspond to p-values

    Journal: bioRxiv

    Article Title: INTS13 Mutations Causing a Developmental Ciliopathy Disrupt Integrator Complex Assembly

    doi: 10.1101/2020.07.20.209130

    Figure Lengend Snippet: Characterization of Loss-of-function INTS13 mutations in patient cells. a RT-qPCR shows that endogenous INTS13 transcript levels were significantly reduced relative to control cells in primary dermal fibroblasts of affected individual II.4 from Family 1. Data are mean +/-SD, n = 3. Statistical significance was calculated using a two-tailed unpaired t-test. b Primary dermal fibroblasts from individual II.4 from Family 1 were treated with 100 g/ml cycloheximide (CHX). Following treatment, RT-qPCR was done which shows that the INTS13 mRNA level was increased in treated cells. Data are mean +/-SEM, n = 3. Statistical significance was calculated using a one-tailed Student’s t-test. c Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 1 using two polyclonal antibodies designed against distinct regions of INTS13. No signal was detected in patient cells with the antibody directed against the C-terminus of INTS13 (C). A truncated protein was detected in patient cells by the antibody directed against the center of the protein (M). Actin was used as a loading control. d RT-qPCR shows that INTS13 transcript levels were significantly reduced in primary dermal fibroblast cells of affected individual II.4 from Family 2 relative to control cells. Data are mean +/-SD, n = 3. Statistical significance was calculated using a two-tailed unpaired t-test. e Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 2 shows reduced levels of INTS13 protein compared to control cells. f Western blot analysis of primary dermal fibroblasts from individual II.4 from Family 2 treated with MG132. Treated cells show a level of INTS13 protein comparable to control cells. * indicates a non-specific signal. g Multiciliated airway cells were obtained from nasal biopsies of three members of Family 1: the unaffected carrier mother (Con.) and the two affected children (II.4 and II.5). Probing for acetylated α-tubulin shows significantly shorter, less dense, and disorganized cilia in the affected individuals compared to the mother’s cells. h Bar graph representing the average length of nasal cilia. Data are mean +/-SEM, n = 200 cells for each individual. Statistical significance was calculated using a one-tailed t-test. *, **, ***, **** correspond to p-values

    Article Snippet: The following antibodies were used for HRP-mediated chemiluminescent: polyclonal Rabbit antibody to INTS13 (C & M), monoclonal Mouse antibody to actin (Chemicon; Mab1501R), β-tubulin (E7, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), dynein heavy chain (P1H4, gift from T. Hays, University of Minnesota, Minneapolis, MN) and anti-HA antibody (Sigma, H 6533 (clone HA-7).

    Techniques: Quantitative RT-PCR, Two Tailed Test, One-tailed Test, Western Blot

    HKU1 neutralization by purified human IVIG and rabbit polyclonal antibodies to the HKU1 spike protein. HKU1 virus was incubated with the antibodies and then inoculated onto HTBE cells at 34 °C for 4 h. Following removal of the inoculum, the cells

    Journal: The Journal of General Virology

    Article Title: Isolation, propagation, genome analysis and epidemiology of HKU1 betacoronaviruses

    doi: 10.1099/vir.0.059832-0

    Figure Lengend Snippet: HKU1 neutralization by purified human IVIG and rabbit polyclonal antibodies to the HKU1 spike protein. HKU1 virus was incubated with the antibodies and then inoculated onto HTBE cells at 34 °C for 4 h. Following removal of the inoculum, the cells

    Article Snippet: HKU1 was detected in infected cells using the rabbit polyclonal antibody directed against the HKU1 spike glycoprotein and visualized using a goat anti-rabbit antibody conjugated to Alexa Fluor 488 (Invitrogen).

    Techniques: Neutralization, Purification, Incubation

    At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.

    Journal: PLoS Pathogens

    Article Title: Chromatin dynamics and the transcriptional competence of HSV-1 genomes during lytic infections

    doi: 10.1371/journal.ppat.1008076

    Figure Lengend Snippet: At after infection, HSV-1 DNA was protected from serial MCN digestion to sizes of mono to poly-nucleosomes that fractionated in complexes with the hydrodynamic ratios of mono to poly-nucleosomes and contain histone H3. Nuclei of infected cells were subjected to serial MCN digestion and then cross-linked. (A) Cross-linked soluble chromatin separated by hydrodynamic ratios in a 0–10% sucrose gradient, and resolved in 2% agarose gel electrophoresis, stained with EtBr. The left pointing arrowheads at the right indicate the migration of mono- to octa-nucleosome-sized DNA. (B) Chromatin immunoprecipitation of HSV-1 DNA with histone H3. EtBr stained agarose gels of saturating PCR-amplified cellular (GAPDH) or HSV-1 (UL25, UL46, ICP4, or gE) DNA co-immunoprecipitated with histone H3. (C) Cartoon presenting the positions of the PCR amplicons in the HSV-1 genome. In, input (2%); H3, anti-histone H3 antibody; IgG, isotype antibody control (labeled only in the No Dig sample for simplicity); No Dig, undigested unfractionated chromatin; Ins, insoluble chromatin.

    Article Snippet: Antibodies against histone H2A (Abcam, rabbit, Cat. ab88770), H2B (Abcam, mouse, Cat. ab52484), H3 (Abcam, rabbit, Cat. ab1791), H4 (Abcam, mouse, Cat. ab31830) were diluted 1:1000 in 10 ml PBS with 0.05% tween-20.

    Techniques: Infection, Agarose Gel Electrophoresis, Staining, Migration, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Amplification, Immunoprecipitation, Labeling

    Set2 is required for Bub1-independent localization of Sgo2 to chromatin. ( A ) ChIP analyses of H3 or H3K36me3 localization in wild-type or set2Δ cells at the log phase or stationary phase. The graph shows the percentage of co-immunoprecipitated DNA with anti-H3 or anti- H3K36me3 antibody per total DNA in whole cell extracts (error bar = range of 2 experiments). ( B ) ChIP analyses of Sgo2-3FLAG localization in WT or set2Δ cells at the log phase (left) or stationary phase (right). The graph shows the percentage of co-immunoprecipitated DNA with anti-FLAG antibody per total DNA in whole cell extracts (error bar = range of 2 experiments).

    Journal: Scientific Reports

    Article Title: Bub1 kinase- and H2A phosphorylation-independent regulation of Shugoshin proteins under glucose-restricted conditions

    doi: 10.1038/s41598-019-39479-6

    Figure Lengend Snippet: Set2 is required for Bub1-independent localization of Sgo2 to chromatin. ( A ) ChIP analyses of H3 or H3K36me3 localization in wild-type or set2Δ cells at the log phase or stationary phase. The graph shows the percentage of co-immunoprecipitated DNA with anti-H3 or anti- H3K36me3 antibody per total DNA in whole cell extracts (error bar = range of 2 experiments). ( B ) ChIP analyses of Sgo2-3FLAG localization in WT or set2Δ cells at the log phase (left) or stationary phase (right). The graph shows the percentage of co-immunoprecipitated DNA with anti-FLAG antibody per total DNA in whole cell extracts (error bar = range of 2 experiments).

    Article Snippet: For H2A, phosphor-H2A, histone H3, and H3-K36me3 IPs, after 150 µL of WCE (1.2 mg/mL) was incubated with anti-H2A (active motif, 39945), anti-H2AS121ph , anti-H3 (abcam, ab1791), anti-H3K36me3 (abcam, ab9050), or normal rabbit IgG (Santa Cruz, sc-2027) antibodies for 30 min on ice, 10 µL of Dynabeads Protein G were added to the WCE-antibody mixture and incubated for 2 hrs at 4 °C.

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation

    EP300 - and CREBBP - ZNF384 fusions resulted in loss of HAT activity, global histone acetylation deregulation, and sensitivity to HDAC inhibition. ( A , B ) HAT enzymatic activity was measured for various EP300 and CREBBP proteins: wild-type, truncated, and

    Journal: Genome Research

    Article Title: Whole-transcriptome sequencing identifies a distinct subtype of acute lymphoblastic leukemia with predominant genomic abnormalities of EP300 and CREBBP

    doi: 10.1101/gr.209163.116

    Figure Lengend Snippet: EP300 - and CREBBP - ZNF384 fusions resulted in loss of HAT activity, global histone acetylation deregulation, and sensitivity to HDAC inhibition. ( A , B ) HAT enzymatic activity was measured for various EP300 and CREBBP proteins: wild-type, truncated, and

    Article Snippet: In transformed Ba/f3 cells, ZNF384 fusion gene expression and histone H3 acetylation were evaluated by Western blot, using anti-acetyl-histone H3 antibody (Millipore, 06-599, 1:1000 dilution), anti-histone H3 antibody (Cell Signaling, 4499S, 1:1,000 dilution), anti-histone H3 (acetyl K9) antibody (Abcam, ab4441, 1:1,000 dilution), and anti-histone H3 (acetyl K27) antibody (Abcam, ab4729, 1:1,000 dilution).

    Techniques: HAT Assay, Activity Assay, Inhibition

    Subcellular localization of WT/APP and 3M/APP by immunofluorescence microscopy. HCN-1A cells (A–L) and COS cells (M–R) were transfected with WT/APP (A–F) or 3M/APP (G–L). The inset in F is a twofold enlargement of a region showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na + / K + ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.

    Journal: The Journal of Cell Biology

    Article Title: Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells

    doi: 10.1083/jcb.200207030

    Figure Lengend Snippet: Subcellular localization of WT/APP and 3M/APP by immunofluorescence microscopy. HCN-1A cells (A–L) and COS cells (M–R) were transfected with WT/APP (A–F) or 3M/APP (G–L). The inset in F is a twofold enlargement of a region showing APP Ab–stained granular structures both overlapping and nonoverlapping with mitochondrial stain, as indicated by arrows. Nonpermeabilized cells (A–C, G–I, and M–O) were double immunostained with APP Nt Ab and monoclonal antibody to Na + / K + ATPase. Permeabilized cells (D–F, J–L, and P–R) were double stained with APP Nt Ab and rabbit polyclonal antibodies to TOM40. Staining patterns (A, B, D, E, G, H, J, K, M, N, P, and Q) were developed with appropriate secondary antibodies conjugated to Alexa dyes. (C, F, I, L, O, and R) Respective overlay patterns.

    Article Snippet: Immunoblots were developed with rabbit polyclonal antibodies directed against the COOH-terminal 22–amino acid sequence of APP (APP Ct Ab), the Aβ Ab (Zymed Laboratories) raised against the 1–40/42 Aβ region of APP, or goat polyclonal antibody against sequence 44–63 of APP (APP Nt Ab) (Calbiochem).

    Techniques: Immunofluorescence, Microscopy, Transfection, Staining

    Interaction of tau with MSI proteins and MSI1 silencing effect on tau levels. (a) Western blot of tau13 in MSI1 IP cytoplasm and nuclear fractions from P301L tau iHEK. (b) Western blot of tau13 in MSI2 IP cytoplasm and nuclear fractions from P301L tau iHEK. (c) Western blot of MSI1 from total lysate of P301L tau iHEK and relative quantification. (d) Western blot of MSI1 from total lysate of WT tau iHEK and relative quantification. (e) Western blot of MSI1, MSI2, and Tau13 in untreated and silenced WT tau iHEK total lysates. (f) MSI1 quantification. (g) MSI2 quantification. (h–i) HMW (75–250 kDa) tau and monomeric form (black arrow) quantification. Bar Graphs are used to show quantification of relative density as function of GAPDH. Student's t test has been use to determine statistical significance. Error bars represent SD

    Journal: Aging Cell

    Article Title: Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration, et al. Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration

    doi: 10.1111/acel.13035

    Figure Lengend Snippet: Interaction of tau with MSI proteins and MSI1 silencing effect on tau levels. (a) Western blot of tau13 in MSI1 IP cytoplasm and nuclear fractions from P301L tau iHEK. (b) Western blot of tau13 in MSI2 IP cytoplasm and nuclear fractions from P301L tau iHEK. (c) Western blot of MSI1 from total lysate of P301L tau iHEK and relative quantification. (d) Western blot of MSI1 from total lysate of WT tau iHEK and relative quantification. (e) Western blot of MSI1, MSI2, and Tau13 in untreated and silenced WT tau iHEK total lysates. (f) MSI1 quantification. (g) MSI2 quantification. (h–i) HMW (75–250 kDa) tau and monomeric form (black arrow) quantification. Bar Graphs are used to show quantification of relative density as function of GAPDH. Student's t test has been use to determine statistical significance. Error bars represent SD

    Article Snippet: Membranes were then probed for 1 hr at room temperature with α‐MSI1 (1:1,000, ab52865 Abcam), α‐MSI2 (1:1,000, ab76148 Abcam), Pan‐Tau (Tau13—1:10,000, MMS‐520R Covance), GAPDH (1:1,000, ab9485 Abcam), LaminB1 (1:1,000, ab133741 Abcam), and Histone3 (1:1,000, ab201456 Abcam) antibodies diluted in 5% nonfat dry milk. α‐MSI1 immunoreactivity and α‐MSI2 immunoreactivity were detected with an HRP‐conjugated anti‐rabbit IgG (1:6,000, GE Healthcare).

    Techniques: Western Blot

    MSI1 and MSI2 in HEK cellular fractions. Western blot of MSI1 in control and treated iHEK WT and iHEK P310L cells. (a) In iHEK WT no significant difference has been observed between cytoplasm (ns, p = .2405) and nuclei (ns, p = .7448) after treatment with Tet. LaminB1 and GAPDH have been used for purity controls for nuclear and cytoplasm fractions, respectively. (b) Western blot of MSI1 in control and treated P301L tau iHEK. As observed in WT cells, MSI1 did not show significant change in the cytoplasm (ns, p = .4573); however, a significant increment has been observed in the nuclei (*** p = .0003). (c) Western blot of MSI2 in control and treated iHEK WT tau did not show significant differences in the cytoplasm (ns, p = .8293) and in the nuclei (ns, p = .1662). (d) In P301L tau iHEK, no significant difference has been observed in the cytoplasm (ns, p = .3755) after treatment with Tet for MSI2 but significantly higher in the nuclei (*** p = .0002), as observed for MSI1. LaminB1 and GAPDH have been used for purity controls for nuclear and cytoplasm fractions, respectively. (e) RT–qPCR of MSI1 in WT iHEK showed increment in gene expression (*** p = .0006). (f) RT–qPCR of MSI2 in WT iHEK showed decrement in gene expression (** p = .0023). (g) RT–qPCR of P301L tau iHEK showed significant increment in MSI1 gene expression (*** p = .0001). (h) RT–qPCR in P301L tau iHEK showed significant increment in MSI2 gene expression (**** p

    Journal: Aging Cell

    Article Title: Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration, et al. Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration

    doi: 10.1111/acel.13035

    Figure Lengend Snippet: MSI1 and MSI2 in HEK cellular fractions. Western blot of MSI1 in control and treated iHEK WT and iHEK P310L cells. (a) In iHEK WT no significant difference has been observed between cytoplasm (ns, p = .2405) and nuclei (ns, p = .7448) after treatment with Tet. LaminB1 and GAPDH have been used for purity controls for nuclear and cytoplasm fractions, respectively. (b) Western blot of MSI1 in control and treated P301L tau iHEK. As observed in WT cells, MSI1 did not show significant change in the cytoplasm (ns, p = .4573); however, a significant increment has been observed in the nuclei (*** p = .0003). (c) Western blot of MSI2 in control and treated iHEK WT tau did not show significant differences in the cytoplasm (ns, p = .8293) and in the nuclei (ns, p = .1662). (d) In P301L tau iHEK, no significant difference has been observed in the cytoplasm (ns, p = .3755) after treatment with Tet for MSI2 but significantly higher in the nuclei (*** p = .0002), as observed for MSI1. LaminB1 and GAPDH have been used for purity controls for nuclear and cytoplasm fractions, respectively. (e) RT–qPCR of MSI1 in WT iHEK showed increment in gene expression (*** p = .0006). (f) RT–qPCR of MSI2 in WT iHEK showed decrement in gene expression (** p = .0023). (g) RT–qPCR of P301L tau iHEK showed significant increment in MSI1 gene expression (*** p = .0001). (h) RT–qPCR in P301L tau iHEK showed significant increment in MSI2 gene expression (**** p

    Article Snippet: Membranes were then probed for 1 hr at room temperature with α‐MSI1 (1:1,000, ab52865 Abcam), α‐MSI2 (1:1,000, ab76148 Abcam), Pan‐Tau (Tau13—1:10,000, MMS‐520R Covance), GAPDH (1:1,000, ab9485 Abcam), LaminB1 (1:1,000, ab133741 Abcam), and Histone3 (1:1,000, ab201456 Abcam) antibodies diluted in 5% nonfat dry milk. α‐MSI1 immunoreactivity and α‐MSI2 immunoreactivity were detected with an HRP‐conjugated anti‐rabbit IgG (1:6,000, GE Healthcare).

    Techniques: Western Blot, Quantitative RT-PCR, Expressing

    WT and P301L tau overexpression in iHEK cell. Western blot of cytoplasmic and nuclear fractions (control and treated (+Tet) cells). Tau species have been revealed using Tau13 Ab in HEK‐293, WT iHEK, and P301L tau iHEK. (a) We observed an increment (as expected) of tau expression and oligomer occurrence in the cytoplasm fractions of WT and P301L tau iHEK. (b) Nuclear tau species, including oligomers, appear after induction with Tet; in the controls, tau is absent (HEK‐293 and iHEK WT) or at low level (P301L tau iHEK). GAPDH and LaminB1 have been used for the purity of cytoplasmic and nuclear extracts, respectively. (c) Representative confocal images of iHEK WT cells, control and treated, stained with Tau13 (1/200, top panel) and T22 (1/300, bottom panel) antibodies, showing increment of signal in treated cells (100× magnification, zoomed 2×, white scale bar: 5 µm), nuclei have been stained with DAPI (blue). (d) Representative confocal images of iHEK P301L tau cells, control and treated, stained with Tau13 (top panel) and T22 (bottom panel) antibodies, showing increment of signal in treated cells (100× magnification, optical zoom 2×, white scale bar: 5 µm), nuclei has been stained with DAPI (blue). On the bottom left of each fluorescence image, the red and blue channels are shown in gray to better observe tau patterns in the cells

    Journal: Aging Cell

    Article Title: Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration, et al. Tau oligomers mediate aggregation of RNA‐binding proteins Musashi1 and Musashi2 inducing Lamin alteration

    doi: 10.1111/acel.13035

    Figure Lengend Snippet: WT and P301L tau overexpression in iHEK cell. Western blot of cytoplasmic and nuclear fractions (control and treated (+Tet) cells). Tau species have been revealed using Tau13 Ab in HEK‐293, WT iHEK, and P301L tau iHEK. (a) We observed an increment (as expected) of tau expression and oligomer occurrence in the cytoplasm fractions of WT and P301L tau iHEK. (b) Nuclear tau species, including oligomers, appear after induction with Tet; in the controls, tau is absent (HEK‐293 and iHEK WT) or at low level (P301L tau iHEK). GAPDH and LaminB1 have been used for the purity of cytoplasmic and nuclear extracts, respectively. (c) Representative confocal images of iHEK WT cells, control and treated, stained with Tau13 (1/200, top panel) and T22 (1/300, bottom panel) antibodies, showing increment of signal in treated cells (100× magnification, zoomed 2×, white scale bar: 5 µm), nuclei have been stained with DAPI (blue). (d) Representative confocal images of iHEK P301L tau cells, control and treated, stained with Tau13 (top panel) and T22 (bottom panel) antibodies, showing increment of signal in treated cells (100× magnification, optical zoom 2×, white scale bar: 5 µm), nuclei has been stained with DAPI (blue). On the bottom left of each fluorescence image, the red and blue channels are shown in gray to better observe tau patterns in the cells

    Article Snippet: Membranes were then probed for 1 hr at room temperature with α‐MSI1 (1:1,000, ab52865 Abcam), α‐MSI2 (1:1,000, ab76148 Abcam), Pan‐Tau (Tau13—1:10,000, MMS‐520R Covance), GAPDH (1:1,000, ab9485 Abcam), LaminB1 (1:1,000, ab133741 Abcam), and Histone3 (1:1,000, ab201456 Abcam) antibodies diluted in 5% nonfat dry milk. α‐MSI1 immunoreactivity and α‐MSI2 immunoreactivity were detected with an HRP‐conjugated anti‐rabbit IgG (1:6,000, GE Healthcare).

    Techniques: Over Expression, Western Blot, Expressing, Staining, Fluorescence

    Ectopic expression of the assigned MmuPV1 ORFs in HEK293 cells. (A) MmuPV1 ORFs and their encoded protein size. (B) Protein sequences of E1^M1 and E1^M2. The N-terminal aa sequences of both E1^M1 and E1^M2 (grey box) are the same as the N-terminal E1 protein, but the N-terminal E1 in the E1^M1 is 23 aa residues longer than E1^M2 (grey box). The C-terminal aa residues of both E1^M1 and E1^M2 are the same and encoded by the same exon 2 being off-frame from E1 and thus totally differ from E1. (C) Expression of individual ORFs in HEK293 cells. Individual ORFs assigned were separately inserted into a mammalian expression vector in frame with a C-terminal FLAG-tag. Expression of the corresponding protein in HEK293 cells was examined by Western blotting using an anti-FLAG antibody at 24 h after transfection. (D) Expressed viral E6 and E7 are labile proteins in HEK293 cells. Cell lysates were prepared from HEK293 cells transfected by an E6 or E7 expression vector in the presence or absence of proteasome inhibitor MG132 for 6 h and examined by Western blotting as in (C). (E) FA staining of E6, E7, E2, E8^E2, E1^E4, E1^M1 and E1^M2 in HeLa cells by anti-FLAG M2 monoclonal antibody in combination with Alexa Flour488-labeled anti-mouse secondary antibody. The cell nuclei were counterstained by Hoechst 33342 dye.

    Journal: PLoS Pathogens

    Article Title: The full transcription map of mouse papillomavirus type 1 (MmuPV1) in mouse wart tissues

    doi: 10.1371/journal.ppat.1006715

    Figure Lengend Snippet: Ectopic expression of the assigned MmuPV1 ORFs in HEK293 cells. (A) MmuPV1 ORFs and their encoded protein size. (B) Protein sequences of E1^M1 and E1^M2. The N-terminal aa sequences of both E1^M1 and E1^M2 (grey box) are the same as the N-terminal E1 protein, but the N-terminal E1 in the E1^M1 is 23 aa residues longer than E1^M2 (grey box). The C-terminal aa residues of both E1^M1 and E1^M2 are the same and encoded by the same exon 2 being off-frame from E1 and thus totally differ from E1. (C) Expression of individual ORFs in HEK293 cells. Individual ORFs assigned were separately inserted into a mammalian expression vector in frame with a C-terminal FLAG-tag. Expression of the corresponding protein in HEK293 cells was examined by Western blotting using an anti-FLAG antibody at 24 h after transfection. (D) Expressed viral E6 and E7 are labile proteins in HEK293 cells. Cell lysates were prepared from HEK293 cells transfected by an E6 or E7 expression vector in the presence or absence of proteasome inhibitor MG132 for 6 h and examined by Western blotting as in (C). (E) FA staining of E6, E7, E2, E8^E2, E1^E4, E1^M1 and E1^M2 in HeLa cells by anti-FLAG M2 monoclonal antibody in combination with Alexa Flour488-labeled anti-mouse secondary antibody. The cell nuclei were counterstained by Hoechst 33342 dye.

    Article Snippet: Total protein extracts and total RNA were prepared 24 h after transfection of HEK293 cells and the expressed individual viral proteins were determined by Western blotting with a rabbit polyclonal anti-FLAG antibody (Sigma-Aldrich, #F7425).

    Techniques: Expressing, Plasmid Preparation, FLAG-tag, Western Blot, Transfection, Staining, Labeling

    Western blot of S100A6, using the polyclonal anti-S100A6 antibody (Dako) on lysates from prostate cancer cell lines. Lane 1. Du145; lane 2, LNCaP; lane 3, PC3. Note the protein expression in Du145 and PC3 cells, seen as an ∼10 kDa band, but its absence in the LNCaP cell line.

    Journal: British Journal of Cancer

    Article Title: S100A6 (Calcyclin) is a prostate basal cell marker absent in prostate cancer and its precursors

    doi: 10.1038/sj.bjc.6602034

    Figure Lengend Snippet: Western blot of S100A6, using the polyclonal anti-S100A6 antibody (Dako) on lysates from prostate cancer cell lines. Lane 1. Du145; lane 2, LNCaP; lane 3, PC3. Note the protein expression in Du145 and PC3 cells, seen as an ∼10 kDa band, but its absence in the LNCaP cell line.

    Article Snippet: The loss of expression seen in the malignant cells was confirmed using two commercially available antibodies, that is, a mouse monoclonal antibody (Sigma) and rabbit polyclonal antibody (Dako).

    Techniques: Western Blot, Expressing

    Depletion of Angpt2 from adipocytes alters subcutaneous fat distribution. a Diagram for generation of Angpt2 i∆Ad mice, inducible and specific deletion of Angpt2 in adipocytes by tamoxifen delivery into 4 week old mice and analyses in 8-week old mice. b Comparisons of Angpt2 mRNA expression in fractionized adipocytes (Ad) of SAT in WT and Angpt2 i∆Ad mice. c , d Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆Ad mice. n = 6 mice/group pooled from three independent experiments. e Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 ∆Ad mice. Four to six different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. f , g Representative images and comparisons of apoptosis (Caspase-3) and beiging (UCP1) in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 50 μm. h , i Representative images and comparison of indicated immune cell infiltration in SAT and VAT of WT and Angpt2 i∆Ad mice. Magnified view is shown in right panels. Scale bars, 30 μm. j , k Representative images and comparisons of vascular density and CD31 + vessel area in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 100 μm. l Diagram for generation of Angpt2 i∆EC mice, inducible and specific deletion of Angpt2 in endothelial cells by tamoxifen delivery into 4 week old mice and analyses in 8 week old mice. m Comparisons of Angpt2 mRNA expression in stromal vascular fraction (SVF) of SAT in WT and Angpt2 i∆EC mice. n , o Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆EC mice. p Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 i∆EC mice. Four different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 3 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two-tailed Student’s t test. NS not significant.

    Journal: Nature Communications

    Article Title: Angiopoietin-2–integrin α5β1 signaling enhances vascular fatty acid transport and prevents ectopic lipid-induced insulin resistance

    doi: 10.1038/s41467-020-16795-4

    Figure Lengend Snippet: Depletion of Angpt2 from adipocytes alters subcutaneous fat distribution. a Diagram for generation of Angpt2 i∆Ad mice, inducible and specific deletion of Angpt2 in adipocytes by tamoxifen delivery into 4 week old mice and analyses in 8-week old mice. b Comparisons of Angpt2 mRNA expression in fractionized adipocytes (Ad) of SAT in WT and Angpt2 i∆Ad mice. c , d Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆Ad mice. n = 6 mice/group pooled from three independent experiments. e Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 ∆Ad mice. Four to six different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. f , g Representative images and comparisons of apoptosis (Caspase-3) and beiging (UCP1) in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 50 μm. h , i Representative images and comparison of indicated immune cell infiltration in SAT and VAT of WT and Angpt2 i∆Ad mice. Magnified view is shown in right panels. Scale bars, 30 μm. j , k Representative images and comparisons of vascular density and CD31 + vessel area in SAT of WT and Angpt2 i∆Ad mice. Scale bars, 100 μm. l Diagram for generation of Angpt2 i∆EC mice, inducible and specific deletion of Angpt2 in endothelial cells by tamoxifen delivery into 4 week old mice and analyses in 8 week old mice. m Comparisons of Angpt2 mRNA expression in stromal vascular fraction (SVF) of SAT in WT and Angpt2 i∆EC mice. n , o Comparison of body weight and fat weight in different adipose tissues between WT and Angpt2 i∆EC mice. p Representative H E-stained images and comparisons in adipocyte size (diameter; μm) of different adipose tissues in WT and Angpt2 i∆EC mice. Four different mice of each genotype were randomly selected to determine the adipocyte size and data are presented as % of total cells. Scale bars, 50 μm. Unless otherwise denoted, each dot indicates a value obtained from one mouse and n = 3 mice/group pooled from two independent experiments. Horizontal bars indicate mean ± SD and P values versus WT by two-tailed Student’s t test. NS not significant.

    Article Snippet: After blocking with 5% goat or donkey serum (Jackson ImmunoResearch) in PBST (0.3% Triton X-100 in PBS for whole-mount method, 0.03% Triton X-100 in PBS for paraffin-sections) for 1 h at RT, whole-mounted or sectioned tissue was incubated overnight at 4 °C with the following primary antibodies (diluted at a ratio of 1:200 in blocking solution): anti-Perilipin (guinea pig polyclonal, 20R-PP004, Fitzgerald), anti-mouse CD31 (hamster monoclonal, 2H8, Millipore), anti-GFP (rabit polycloncal, AB3080, Millipore), anti-cleaved caspase-3 (rabbit polyclonal, 9661, Cell Signaling), anti-UCP1 (rabit polycloncal, ab23841, Abcam), anti-Integrin α5β1 (rat monoclonal, BMB5, Millipore), anti- active Integrin β1 (rat monoclonal, 9EG7, BD Biosciences), anti- Integrin β1 (mouse monoclonal, 12G10, Abcam), anti-HA (rabbit polyclonal, H6908, Sigma-Aldrich), and anti-human CD31 (rabit polyclonal, ab28364, Abcam).

    Techniques: Mouse Assay, Expressing, Staining, Two Tailed Test

    Sexually dimorphic Mip neurons in the AG that regulate female receptivity. a Mip neurons in the AG from a female (above left) and a male (above right) carrying Mip-GAL4 , Mip6.0-GAL80 and UAS-mCD8-GFP stained by anti-GFP (green) and anti-nc82 (magenta) antibodies. The lower panels indicate the numbers and relative locations of Mip neuron somas in female (left) and male (right) AGs. The Mip neurons in the AG are grouped into six subsets: medial anterior lateral ( mAL ), ventral anterior lateral ( vAL ), ventral anterior medial ( vAM ), small ventral posterior medial ( s-vPM ), large ventral posterior medial ( l-vPM ), and small medial posterior medial ( s-mPM ). Filled and open circles indicate neurons positive and negative for anti-Mip, repectively. Scale bars, 50 μm. b The numbers of GFP-positive Mip neurons in the AGs of females (green) and males (red) carrying Mip-GAL4 , Mip6.0-GAL80 and UAS-mCD8-EGFP . Note the clear sexual dimorphism in the vAL and vAM neurons. c Negative images of TRIC labelling (anti-GFP) in the AGs of virgin (upper panels) and mated females (lower panels), indicating intracellular Ca 2+ transients. Scale bars, 10 μm. d The GFP intensities from vAM , l-vPM and SAG neurons of TRIC females show Ca 2+ activity in virgin (green) and mated females (red). e The frequencies of labelled (and therefore activated) neurons in the indicated Mip neuron subset of the AG from non-re-mating (green) and re-mating mosaic females (red). We used females carrying hsFLP , Mip-GAL4 , UAS-TrpA1 , UAS-DsRed and Tub FRT GAL80 FRT for stochastic manipulation (for details, see the text). NS indicates non-significance ( P > 0.05); ** P

    Journal: Nature Communications

    Article Title: Female-specific myoinhibitory peptide neurons regulate mating receptivity in Drosophila melanogaster

    doi: 10.1038/s41467-017-01794-9

    Figure Lengend Snippet: Sexually dimorphic Mip neurons in the AG that regulate female receptivity. a Mip neurons in the AG from a female (above left) and a male (above right) carrying Mip-GAL4 , Mip6.0-GAL80 and UAS-mCD8-GFP stained by anti-GFP (green) and anti-nc82 (magenta) antibodies. The lower panels indicate the numbers and relative locations of Mip neuron somas in female (left) and male (right) AGs. The Mip neurons in the AG are grouped into six subsets: medial anterior lateral ( mAL ), ventral anterior lateral ( vAL ), ventral anterior medial ( vAM ), small ventral posterior medial ( s-vPM ), large ventral posterior medial ( l-vPM ), and small medial posterior medial ( s-mPM ). Filled and open circles indicate neurons positive and negative for anti-Mip, repectively. Scale bars, 50 μm. b The numbers of GFP-positive Mip neurons in the AGs of females (green) and males (red) carrying Mip-GAL4 , Mip6.0-GAL80 and UAS-mCD8-EGFP . Note the clear sexual dimorphism in the vAL and vAM neurons. c Negative images of TRIC labelling (anti-GFP) in the AGs of virgin (upper panels) and mated females (lower panels), indicating intracellular Ca 2+ transients. Scale bars, 10 μm. d The GFP intensities from vAM , l-vPM and SAG neurons of TRIC females show Ca 2+ activity in virgin (green) and mated females (red). e The frequencies of labelled (and therefore activated) neurons in the indicated Mip neuron subset of the AG from non-re-mating (green) and re-mating mosaic females (red). We used females carrying hsFLP , Mip-GAL4 , UAS-TrpA1 , UAS-DsRed and Tub FRT GAL80 FRT for stochastic manipulation (for details, see the text). NS indicates non-significance ( P > 0.05); ** P

    Article Snippet: Antibodies used were: rabbit anti-GFP (1:1000; Invitrogen, A11122), mouse anti-GFP (1:1000; Sigma, G6539), rabbit anti-DsRed (1:1000; Clontech, 632496), mouse anti-nc82 (1:50; Developmental Studies Hybridoma Bank), Alexa 488-conjugated goat anti-mouse (1:1000; Invitrogen, A11001), Alexa 488-conjugated goat anti-rabbit (1:1000; Invitrogen, A11008), Alexa 568-conjugated goat anti-mouse (1:1000; Invitrogen, A11004), and Alexa 568-conjugated goat anti-rabbit (1:1000; Invitrogen, A11011).

    Techniques: Staining, Activity Assay