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    Structured Review

    Millipore polyclonal antibodies
    Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with <t>polyclonal</t> antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.
    Polyclonal Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy"

    Article Title: ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007605

    Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.
    Figure Legend Snippet: Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.

    Techniques Used: Western Blot, Sequencing, CRISPR, Staining

    2) Product Images from "The Locus Ceruleus Responds to Signaling Molecules Obtained from the CSF by Transfer through Tanycytes"

    Article Title: The Locus Ceruleus Responds to Signaling Molecules Obtained from the CSF by Transfer through Tanycytes

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.5018-10.2011

    Both endogenous and exogenous NGF in the CSF regulate the nuclear sizes of LoC neurons. A , Dot blot experiments show that our polyclonal neutralizing NGF antibody specifically recognizes NGF with minimal cross-reactivity for BDNF and NT-3. B–D
    Figure Legend Snippet: Both endogenous and exogenous NGF in the CSF regulate the nuclear sizes of LoC neurons. A , Dot blot experiments show that our polyclonal neutralizing NGF antibody specifically recognizes NGF with minimal cross-reactivity for BDNF and NT-3. B–D

    Techniques Used: Dot Blot

    3) Product Images from "Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities"

    Article Title: Mutant SOD1 in neuronal mitochondria causes toxicity and mitochondrial dynamics abnormalities

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddp421

    Expression and localization of IMS-targeted hSOD1 in NSC34 cells. ( A ) Western blot of cellular homogenates from cells stably expressing WT and G93A IMS-hSOD1 using a polyclonal anti-SOD1 antibody that recognizes both the human and the mouse protein. mSOD1,
    Figure Legend Snippet: Expression and localization of IMS-targeted hSOD1 in NSC34 cells. ( A ) Western blot of cellular homogenates from cells stably expressing WT and G93A IMS-hSOD1 using a polyclonal anti-SOD1 antibody that recognizes both the human and the mouse protein. mSOD1,

    Techniques Used: Expressing, Western Blot, Stable Transfection

    4) Product Images from "Role of mammary epithelial and stromal P450 enzymes in the clearance and metabolic activation of 7,12-dimethylbenz(a)anthracene in mice"

    Article Title: Role of mammary epithelial and stromal P450 enzymes in the clearance and metabolic activation of 7,12-dimethylbenz(a)anthracene in mice

    Journal: Toxicology letters

    doi: 10.1016/j.toxlet.2012.05.005

    Immunohistochemical and immunoblot analyses of CPR protein expression in the mammary gland of MEpi-Cpr-null and WT mice A. Paraffin sections (4 μm) of mammary gland from female virgin (2-month old) WT and MEpi-Cpr-null mice were analyzed by immunohistochemistry. The tissue sections were incubated with a polyclonal rabbit anti-rat CPR antibody. Alexa Fluor 594 Tyramide Signal Amplification Kit was used for visualization of CPR expression sites (Red). The nucleus was stained with DAPI (Blue). No signal was detected when the primary antibody was replaced by a normal rabbit serum (data not shown). The arrow indicates the epithelial cells. Results shown are typical of six mice per strain analyzed. Scale bar, 20 μm. B. Microsomal proteins (10 μg per lane) from pooled mammary glands of three WT or three MEpi-Cpr-null mice were analyzed for CPR expression with a rabbit anti-rat CPR antibody. Three different microsomal samples were analyzed for each strain. C. Densitometry analysis of the immunoblot results. Values represent means ± S.D., n=3. There was no significant difference in CPR band intensity between MEpi-Cpr-null and WT mice.
    Figure Legend Snippet: Immunohistochemical and immunoblot analyses of CPR protein expression in the mammary gland of MEpi-Cpr-null and WT mice A. Paraffin sections (4 μm) of mammary gland from female virgin (2-month old) WT and MEpi-Cpr-null mice were analyzed by immunohistochemistry. The tissue sections were incubated with a polyclonal rabbit anti-rat CPR antibody. Alexa Fluor 594 Tyramide Signal Amplification Kit was used for visualization of CPR expression sites (Red). The nucleus was stained with DAPI (Blue). No signal was detected when the primary antibody was replaced by a normal rabbit serum (data not shown). The arrow indicates the epithelial cells. Results shown are typical of six mice per strain analyzed. Scale bar, 20 μm. B. Microsomal proteins (10 μg per lane) from pooled mammary glands of three WT or three MEpi-Cpr-null mice were analyzed for CPR expression with a rabbit anti-rat CPR antibody. Three different microsomal samples were analyzed for each strain. C. Densitometry analysis of the immunoblot results. Values represent means ± S.D., n=3. There was no significant difference in CPR band intensity between MEpi-Cpr-null and WT mice.

    Techniques Used: Immunohistochemistry, Expressing, Mouse Assay, Incubation, Amplification, Staining

    5) Product Images from "Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag"

    Article Title: Differential control of CXCR4 and CD4 downregulation by HIV-1 Gag

    Journal: Virology Journal

    doi: 10.1186/1743-422X-5-23

    HIV-1 Gag attenuates SDF-induced CXCR4 downregulation in Jurkat T cells . (A) Jurkat T cells were pre-treated with cycloheximide, then incubated in the presence (filled squares) or absence (open squares) of SDF, PMA and ionomycin for the indicated times. At each time point, cells were lysed and analysed by SDS-PAGE and Western blotting with an anti-CXCR4 polyclonal antibody. Western blots were quantitated and amount of CXCR4 remaining at each time point was determined as a percent of amount of CXCR4 at 0 hour. Data from one representative experiment (out of three) is shown. (B) Jurkat T cells were transduced with Gag-GFP encoding lentiviruses at the indicated MOIs. 48 hours post transduction, cells were analyzed for GFP fluorescence by flow cytometry. The % of GFP positive cells is indicated for each MOI. (C) A representative gel depicting CXCR4 levels in Jurkat T cells transduced with lentiviruses encoding wild-type Gag-GFP and treated as described in (A) is shown. Control represents untransduced cells. (D) Quantitation of the gel shown in (C). Additionally, degradation of CXCR4 in Jurkat T cells transduced with lentiviruses encoding LTAL Gag-GFP is shown. Error bars represent standard deviation between duplicates at each time point. (E) Jurkat T cells were transduced with lentivirus encoding LacZ (control), Gag-GFP or LTAL Gag-GFP. Data shown represents the mean ± SD of % undegraded CXCR4 remaining after 6 hours of incubation with SDF, PMA and ionomycin. (F) Cell surface levels of CXCR4 in Jurkat T cells expressing LacZ, Gag-GFP or LTAL Gag-GFP were determined 48 hours post transduction by flow cytometric analysis of cells stained with a biotinylated anti-CXCR4 antibody and Streptavidin-PE. Data shown represents the mean ± SD of surface CXCR4 levels relative to the control, from two independent experiments.
    Figure Legend Snippet: HIV-1 Gag attenuates SDF-induced CXCR4 downregulation in Jurkat T cells . (A) Jurkat T cells were pre-treated with cycloheximide, then incubated in the presence (filled squares) or absence (open squares) of SDF, PMA and ionomycin for the indicated times. At each time point, cells were lysed and analysed by SDS-PAGE and Western blotting with an anti-CXCR4 polyclonal antibody. Western blots were quantitated and amount of CXCR4 remaining at each time point was determined as a percent of amount of CXCR4 at 0 hour. Data from one representative experiment (out of three) is shown. (B) Jurkat T cells were transduced with Gag-GFP encoding lentiviruses at the indicated MOIs. 48 hours post transduction, cells were analyzed for GFP fluorescence by flow cytometry. The % of GFP positive cells is indicated for each MOI. (C) A representative gel depicting CXCR4 levels in Jurkat T cells transduced with lentiviruses encoding wild-type Gag-GFP and treated as described in (A) is shown. Control represents untransduced cells. (D) Quantitation of the gel shown in (C). Additionally, degradation of CXCR4 in Jurkat T cells transduced with lentiviruses encoding LTAL Gag-GFP is shown. Error bars represent standard deviation between duplicates at each time point. (E) Jurkat T cells were transduced with lentivirus encoding LacZ (control), Gag-GFP or LTAL Gag-GFP. Data shown represents the mean ± SD of % undegraded CXCR4 remaining after 6 hours of incubation with SDF, PMA and ionomycin. (F) Cell surface levels of CXCR4 in Jurkat T cells expressing LacZ, Gag-GFP or LTAL Gag-GFP were determined 48 hours post transduction by flow cytometric analysis of cells stained with a biotinylated anti-CXCR4 antibody and Streptavidin-PE. Data shown represents the mean ± SD of surface CXCR4 levels relative to the control, from two independent experiments.

    Techniques Used: Incubation, SDS Page, Western Blot, Transduction, Fluorescence, Flow Cytometry, Cytometry, Quantitation Assay, Standard Deviation, Expressing, Staining

    6) Product Images from "Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency"

    Article Title: Regulation of epithelial sodium channels in urokinase plasminogen activator deficiency

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00126.2014

    Proteolysis of native ENaC proteins by uPA. A : characterization of a new polyclonal antibody against the COOH-terminal peptide of rat γ ENaC. Both total (γ FLAG T) and biotinylated plasma membrane proteins (γ FLAG M) from Xenopus oocytes
    Figure Legend Snippet: Proteolysis of native ENaC proteins by uPA. A : characterization of a new polyclonal antibody against the COOH-terminal peptide of rat γ ENaC. Both total (γ FLAG T) and biotinylated plasma membrane proteins (γ FLAG M) from Xenopus oocytes

    Techniques Used:

    7) Product Images from "Using Proteomic Approach to Identify Tumor-Associated Proteins as Biomarkers in Human Esophageal Squamous Cell Carcinoma"

    Article Title: Using Proteomic Approach to Identify Tumor-Associated Proteins as Biomarkers in Human Esophageal Squamous Cell Carcinoma

    Journal: Journal of proteome research

    doi: 10.1021/pr200141c

    (a) Expression of HSP70 in ESCC tissues examined by IHC. Tissue-array slide was stained with polyclonal anti-HSP70 antibody at a 1:500 dilution. A: A representative normal esophagus tissue was negatively stained with anti-HSP70 antibody; C, E and G: Representative
    Figure Legend Snippet: (a) Expression of HSP70 in ESCC tissues examined by IHC. Tissue-array slide was stained with polyclonal anti-HSP70 antibody at a 1:500 dilution. A: A representative normal esophagus tissue was negatively stained with anti-HSP70 antibody; C, E and G: Representative

    Techniques Used: Expressing, Immunohistochemistry, Staining

    8) Product Images from "The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype"

    Article Title: The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039097

    rLCC111 substrates do not support cell attachment. Adhesion of HT0180 cells to plastic-coated LM111 or rLCC111 (ranging from 0.08 to 20 μg/ml), or BSA was measured by bioluminescence. (A). Data are plotted as the log of fold change in adhesion relative to BSA. The coating efficiency of LM111 and rLCC111 was monitored by ELISA using a polyclonal anti-laminin antibody (B). Soluble rLCC111 (5 to 20 mg/ml) inhibits adhesion of HT1080 cells (C) to plastic immobilized intact LM111 (10 mg/ml). Data shown are from a representative experiment out of three independent ones. (*, p
    Figure Legend Snippet: rLCC111 substrates do not support cell attachment. Adhesion of HT0180 cells to plastic-coated LM111 or rLCC111 (ranging from 0.08 to 20 μg/ml), or BSA was measured by bioluminescence. (A). Data are plotted as the log of fold change in adhesion relative to BSA. The coating efficiency of LM111 and rLCC111 was monitored by ELISA using a polyclonal anti-laminin antibody (B). Soluble rLCC111 (5 to 20 mg/ml) inhibits adhesion of HT1080 cells (C) to plastic immobilized intact LM111 (10 mg/ml). Data shown are from a representative experiment out of three independent ones. (*, p

    Techniques Used: Cell Attachment Assay, Enzyme-linked Immunosorbent Assay

    9) Product Images from "CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast"

    Article Title: CLN1 and Its Repression by Xbp1 Are Important for Efficient Sporulation in Budding Yeast

    Journal: Molecular and Cellular Biology

    doi:

    Expression of XBP1 during meiosis. An SK-1 derivative strain (NKY 278) was shifted at time zero from presporulation medium (YEPA) to sporulation medium. Progression through meiosis was monitored, and aliquots were taken every 2 h for up to 16 h. After 24 h, the culture was shifted to rich medium (YEPD) and monitored for another 4 h. (A) Progression through meiosis was monitored by measuring the DNA content at the indicated time points by FACS analysis and analyzing the data with the CellQuest program. The positions of peaks representing 2n or 4n DNA content are labeled. (B) Total RNA was prepared, and the expression of XBP1 , ACT1 , DMC1 , NDT80 , SGA1 , DIT1 , and SPS100 was analyzed by Northern blotting. The blots were hybridized simultaneously or sequentially. The ethidium bromide (EtBr)-stained gel is shown at the bottom. Exposure times were 8 h for the XBP1 -specific hybridization and 1 h for the SGA1 -specific hybridization. (C) Protein extracts were prepared from the same time points and subjected to immunoprecipitation using a polyclonal antibody directed against Xbp1. Precipitated proteins were analyzed by Western blotting using the same antibody. The 72-kDa Xbp1 band is shown.
    Figure Legend Snippet: Expression of XBP1 during meiosis. An SK-1 derivative strain (NKY 278) was shifted at time zero from presporulation medium (YEPA) to sporulation medium. Progression through meiosis was monitored, and aliquots were taken every 2 h for up to 16 h. After 24 h, the culture was shifted to rich medium (YEPD) and monitored for another 4 h. (A) Progression through meiosis was monitored by measuring the DNA content at the indicated time points by FACS analysis and analyzing the data with the CellQuest program. The positions of peaks representing 2n or 4n DNA content are labeled. (B) Total RNA was prepared, and the expression of XBP1 , ACT1 , DMC1 , NDT80 , SGA1 , DIT1 , and SPS100 was analyzed by Northern blotting. The blots were hybridized simultaneously or sequentially. The ethidium bromide (EtBr)-stained gel is shown at the bottom. Exposure times were 8 h for the XBP1 -specific hybridization and 1 h for the SGA1 -specific hybridization. (C) Protein extracts were prepared from the same time points and subjected to immunoprecipitation using a polyclonal antibody directed against Xbp1. Precipitated proteins were analyzed by Western blotting using the same antibody. The 72-kDa Xbp1 band is shown.

    Techniques Used: Expressing, FACS, Labeling, Northern Blot, Staining, Hybridization, Immunoprecipitation, Western Blot

    10) Product Images from "Protein nanoparticles with ligand-binding and enzymatic activities"

    Article Title: Protein nanoparticles with ligand-binding and enzymatic activities

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S177627

    Results of binding of protein NP with specific ligands. Notes: ( A ) Scheme of interaction of BSA-RhoB NP with immobilized anti-BSA polyclonal antibodies. ( B ) Data of immunofluorescence assay according to scheme A. The error bars correspond to standard deviations (±SD). Differences between BSA-RhoB and corresponding NP were not statistically significant ( P .0.05). ( C ) Scheme of interaction of NP from monoclonal antibodies against HBsAg with the immobilized recombinant antigen. ( D ) ELISA data according to the scheme C. Abbreviations: HBSAg, hepatitis B virus S antigen; BSA, bovine serum albumin; NP, nanoparticles; Mab, monoclonal antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; o.u, optical units; RhoB, fluorescent rhodamine B dye; RFU, relative fluorescent units.
    Figure Legend Snippet: Results of binding of protein NP with specific ligands. Notes: ( A ) Scheme of interaction of BSA-RhoB NP with immobilized anti-BSA polyclonal antibodies. ( B ) Data of immunofluorescence assay according to scheme A. The error bars correspond to standard deviations (±SD). Differences between BSA-RhoB and corresponding NP were not statistically significant ( P .0.05). ( C ) Scheme of interaction of NP from monoclonal antibodies against HBsAg with the immobilized recombinant antigen. ( D ) ELISA data according to the scheme C. Abbreviations: HBSAg, hepatitis B virus S antigen; BSA, bovine serum albumin; NP, nanoparticles; Mab, monoclonal antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; o.u, optical units; RhoB, fluorescent rhodamine B dye; RFU, relative fluorescent units.

    Techniques Used: Binding Assay, Immunofluorescence, Recombinant, Enzyme-linked Immunosorbent Assay

    11) Product Images from "Fibronectin Facilitates Mycobacterium tuberculosis Attachment to Murine Alveolar Macrophages"

    Article Title: Fibronectin Facilitates Mycobacterium tuberculosis Attachment to Murine Alveolar Macrophages

    Journal: Infection and Immunity

    doi: 10.1128/IAI.70.3.1287-1292.2002

    Effects of anti-Fn polyclonal antibodies and MAbs on in vitro attachment of M. tuberculosis to AMs. Fn-enhanced attachment of M. tuberculosis to AMs was examined in the presence of Fn and in the presence or absence of anti-Fn polyclonal antibodies and MAbs directed against the CBD, HBD, and GBD of Fn. Anti-Fn polyclonal antibody significantly decreased the Fn-enhanced attachment of M. tuberculosis to AMs ( P
    Figure Legend Snippet: Effects of anti-Fn polyclonal antibodies and MAbs on in vitro attachment of M. tuberculosis to AMs. Fn-enhanced attachment of M. tuberculosis to AMs was examined in the presence of Fn and in the presence or absence of anti-Fn polyclonal antibodies and MAbs directed against the CBD, HBD, and GBD of Fn. Anti-Fn polyclonal antibody significantly decreased the Fn-enhanced attachment of M. tuberculosis to AMs ( P

    Techniques Used: In Vitro, Affinity Magnetic Separation

    12) Product Images from "Matrix Metalloproteinase-9-Dependent Exposure of a Cryptic Migratory Control Site in Collagen is Required before Retinal Angiogenesis"

    Article Title: Matrix Metalloproteinase-9-Dependent Exposure of a Cryptic Migratory Control Site in Collagen is Required before Retinal Angiogenesis

    Journal: The American Journal of Pathology

    doi:

    Exposure of HUIV26 cryptic sites in MMP-deficient mice. Retinal angiogenesis was induced in MMP-deficient mice and wild-type controls. Retinas from P15 mice were co-stained with biotinylated mAb HUIV26 and either FITC- L. esculentum lectin or polyclonal antibody to collagen type IV. The total number of HUIV26 cryptic sites (spots) was manually counted. Examples of co-stained retinas from MMP-2-null (MMP-2−/−) and wild-type (MMP-2+/+) mice, as well as MMP-9-null (MMP-9−/−) and wild-type (MMP-9+/+) mice. Red indicates exposure of the HUIV26 cryptic sites and green indicates endothelial cell staining with FITC- L. esculentum lectin. Yellow indicates co-localization. White bars represent 50 μm.
    Figure Legend Snippet: Exposure of HUIV26 cryptic sites in MMP-deficient mice. Retinal angiogenesis was induced in MMP-deficient mice and wild-type controls. Retinas from P15 mice were co-stained with biotinylated mAb HUIV26 and either FITC- L. esculentum lectin or polyclonal antibody to collagen type IV. The total number of HUIV26 cryptic sites (spots) was manually counted. Examples of co-stained retinas from MMP-2-null (MMP-2−/−) and wild-type (MMP-2+/+) mice, as well as MMP-9-null (MMP-9−/−) and wild-type (MMP-9+/+) mice. Red indicates exposure of the HUIV26 cryptic sites and green indicates endothelial cell staining with FITC- L. esculentum lectin. Yellow indicates co-localization. White bars represent 50 μm.

    Techniques Used: Mouse Assay, Staining

    Time course of expression of collagen type IV, VEGF, and MMP-9 in the ischemic retina. Ischemia-induced retinal neovascularization was initiated and retinas subjected to hyperoxia were obtained at 0, 1, 2, 3, and 5 days (P12, P13, P14, P15, and P17) after their return to normoxic conditions. A: Retinas were co-stained with a polyclonal antibody directed to collagen type IV (green) and biotinylated mAb HUIV26 (red). B: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and a mAb directed to VEGF (red). C: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and biotinylated mAb HUIV26 (red). D: Retinas were co-stained with a polyclonal antibody directed to MMP-2 (green) and biotinylated mAb HUIV26 (red). Flat mount retinas were analyzed on a Nikon TE300 microscope with epifluoresence filters and images were captured with a charge-couple device camera for analysis. Co-localization is indicated in yellow. White bars equal 50 μm. E: Quantitation of the number of HUIV26 cryptic sites (spots) per retina. The total number of HUIV26 cryptic sites (spots) was manually counted within the total area of each retina and 18 retinas were counted per time point. Data bars represent the mean ± SD.
    Figure Legend Snippet: Time course of expression of collagen type IV, VEGF, and MMP-9 in the ischemic retina. Ischemia-induced retinal neovascularization was initiated and retinas subjected to hyperoxia were obtained at 0, 1, 2, 3, and 5 days (P12, P13, P14, P15, and P17) after their return to normoxic conditions. A: Retinas were co-stained with a polyclonal antibody directed to collagen type IV (green) and biotinylated mAb HUIV26 (red). B: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and a mAb directed to VEGF (red). C: Retinas were co-stained with a polyclonal antibody directed to MMP-9 (green) and biotinylated mAb HUIV26 (red). D: Retinas were co-stained with a polyclonal antibody directed to MMP-2 (green) and biotinylated mAb HUIV26 (red). Flat mount retinas were analyzed on a Nikon TE300 microscope with epifluoresence filters and images were captured with a charge-couple device camera for analysis. Co-localization is indicated in yellow. White bars equal 50 μm. E: Quantitation of the number of HUIV26 cryptic sites (spots) per retina. The total number of HUIV26 cryptic sites (spots) was manually counted within the total area of each retina and 18 retinas were counted per time point. Data bars represent the mean ± SD.

    Techniques Used: Expressing, Staining, Microscopy, Quantitation Assay

    13) Product Images from "The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection"

    Article Title: The Autotransporter BpaB Contributes to the Virulence of Burkholderia mallei in an Aerosol Model of Infection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126437

    BpaB production by E . coli recombinant strains. Panel A : Equivalent amounts of whole cell lysates (WCL), total membrane proteins (TMP) and sarkosyl-insoluble fractions containing OM proteins (OMP) were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with the monoclonal antibody BpaB-MAb#4. Molecular weight markers are shown to the left in kilodaltons. Panel B : Non-permeabilized E . coli strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) and with α-BpaB polyclonal antibodies (green) as described in Materials and Methods. Bacteria were visualized by microscopy using a Nikon Eclipse Ti confocal system. Representative microscopic fields are shown. Panel C : Non-permeabilized E . coli strains were incubated with polyclonal antibodies against BpaB and fluorescently-labeled with a goat α-mouse antibody conjugated with the fluorochrome Alexa Fluor 488. Labeled bacteria were analyzed using a BD LSR II flow cytometer. The x -axis represents the level of fluorescence, and the y -axis corresponds to the particles counted in arbitrary units. The number of cells analyzed, and the percentage of those producing BpaB on their surface, is shown.
    Figure Legend Snippet: BpaB production by E . coli recombinant strains. Panel A : Equivalent amounts of whole cell lysates (WCL), total membrane proteins (TMP) and sarkosyl-insoluble fractions containing OM proteins (OMP) were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with the monoclonal antibody BpaB-MAb#4. Molecular weight markers are shown to the left in kilodaltons. Panel B : Non-permeabilized E . coli strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) and with α-BpaB polyclonal antibodies (green) as described in Materials and Methods. Bacteria were visualized by microscopy using a Nikon Eclipse Ti confocal system. Representative microscopic fields are shown. Panel C : Non-permeabilized E . coli strains were incubated with polyclonal antibodies against BpaB and fluorescently-labeled with a goat α-mouse antibody conjugated with the fluorochrome Alexa Fluor 488. Labeled bacteria were analyzed using a BD LSR II flow cytometer. The x -axis represents the level of fluorescence, and the y -axis corresponds to the particles counted in arbitrary units. The number of cells analyzed, and the percentage of those producing BpaB on their surface, is shown.

    Techniques Used: Recombinant, SDS Page, Western Blot, Molecular Weight, Labeling, Microscopy, Incubation, Flow Cytometry, Cytometry, Fluorescence

    BpaB production by B . mallei recombinant and WT strains. Panel A : Equivalent amounts of whole cell lysate preparations were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with BpaB-MAb#4. Molecular weight markers are shown to the left in kilodaltons. Panel B : Non-permeabilized B . mallei strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) an d with α-BpaB polyclonal antibodies (green) as described in Materials and Methods. Bacteria were visualized by microscopy using a Nikon Eclipse Ti confocal system. Representative microscopic fields are shown. Panel C : Paraformaldehyde-fixed B . mallei strains were incubated with polyclonal antibodies against BpaB and fluorescently-labeled with a goat α-mouse antibody conjugated with the fluorochrome Alexa Fluor 488. Labeled bacteria were analyzed using a BD LSR II flow cytometer. The x -axis represents the level of fluorescence, and the y -axis corresponds to the particles counted in arbitrary units. The number of cells analyzed, and the percentage of those producing BpaB on their surface, is shown.
    Figure Legend Snippet: BpaB production by B . mallei recombinant and WT strains. Panel A : Equivalent amounts of whole cell lysate preparations were resolved by SDS-PAGE, transferred to PVDF membranes and analyzed by western blot with BpaB-MAb#4. Molecular weight markers are shown to the left in kilodaltons. Panel B : Non-permeabilized B . mallei strains were fixed onto glass slides and fluorescently-labeled with DAPI (blue) an d with α-BpaB polyclonal antibodies (green) as described in Materials and Methods. Bacteria were visualized by microscopy using a Nikon Eclipse Ti confocal system. Representative microscopic fields are shown. Panel C : Paraformaldehyde-fixed B . mallei strains were incubated with polyclonal antibodies against BpaB and fluorescently-labeled with a goat α-mouse antibody conjugated with the fluorochrome Alexa Fluor 488. Labeled bacteria were analyzed using a BD LSR II flow cytometer. The x -axis represents the level of fluorescence, and the y -axis corresponds to the particles counted in arbitrary units. The number of cells analyzed, and the percentage of those producing BpaB on their surface, is shown.

    Techniques Used: Recombinant, SDS Page, Western Blot, Molecular Weight, Labeling, Microscopy, Incubation, Flow Cytometry, Cytometry, Fluorescence

    14) Product Images from "Mff functions with Pex11p? and DLP1 in peroxisomal fission"

    Article Title: Mff functions with Pex11p? and DLP1 in peroxisomal fission

    Journal: Biology Open

    doi: 10.1242/bio.20135298

    Mff is localized to peroxisomes and mitochondria. ( A ) The domain structure of human Mff splicing variant 8 is presented. The red, blue, and green boxes indicate the two repeat regions, coiled-coil domain and TMD, respectively. The N-terminal 27–173 amino acid portion of human Mff splicing variant 8 was used as an antigen to raise rabbit anti-Mff polyclonal antibody. ( B ) Cytosol and organelle fractions prepared from HeLa, HEK293, and CHO-K1 cells were analyzed by SDS-PAGE and immunoblotting using antibodies to Mff and Tom20. ( C ) HEK293 cells were treated for 72 h with two different dsRNAs ( MFF #1 and MFF #2). Mff levels were assessed by immunoblotting with anti-Mff antibody. Actin was used as a loading control. ( D ) Control fibroblasts were stained with antibodies to Mff ( a ), Pex14p ( b ), and Tom20 ( c ); the merged view of the three proteins is shown ( d ). Scale bar: 10 µm. Insets, higher magnification images of the boxed regions, scale bar: 2 µm. ( E ) PHM fraction from control fibroblasts was fractionated by Opti-prep density gradient ultracentrifugation. The distribution of peroxisomes, mitochondria, and smooth ER was assessed by immunoblotting using antibodies to the marker proteins Pex14p, Tom20, and P450 reductase (P450r), respectively. Downward solid arrowheads indicate the peak fractions of peroxisomes; the upward open arrowhead indicates Mff (Ps; lane 12 and 13).
    Figure Legend Snippet: Mff is localized to peroxisomes and mitochondria. ( A ) The domain structure of human Mff splicing variant 8 is presented. The red, blue, and green boxes indicate the two repeat regions, coiled-coil domain and TMD, respectively. The N-terminal 27–173 amino acid portion of human Mff splicing variant 8 was used as an antigen to raise rabbit anti-Mff polyclonal antibody. ( B ) Cytosol and organelle fractions prepared from HeLa, HEK293, and CHO-K1 cells were analyzed by SDS-PAGE and immunoblotting using antibodies to Mff and Tom20. ( C ) HEK293 cells were treated for 72 h with two different dsRNAs ( MFF #1 and MFF #2). Mff levels were assessed by immunoblotting with anti-Mff antibody. Actin was used as a loading control. ( D ) Control fibroblasts were stained with antibodies to Mff ( a ), Pex14p ( b ), and Tom20 ( c ); the merged view of the three proteins is shown ( d ). Scale bar: 10 µm. Insets, higher magnification images of the boxed regions, scale bar: 2 µm. ( E ) PHM fraction from control fibroblasts was fractionated by Opti-prep density gradient ultracentrifugation. The distribution of peroxisomes, mitochondria, and smooth ER was assessed by immunoblotting using antibodies to the marker proteins Pex14p, Tom20, and P450 reductase (P450r), respectively. Downward solid arrowheads indicate the peak fractions of peroxisomes; the upward open arrowhead indicates Mff (Ps; lane 12 and 13).

    Techniques Used: Variant Assay, SDS Page, Staining, Marker

    15) Product Images from "Protein nanoparticles with ligand-binding and enzymatic activities"

    Article Title: Protein nanoparticles with ligand-binding and enzymatic activities

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S177627

    Results of binding of protein NP with specific ligands. Notes: ( A ) Scheme of interaction of BSA-RhoB NP with immobilized anti-BSA polyclonal antibodies. ( B ) Data of immunofluorescence assay according to scheme A. The error bars correspond to standard deviations (±SD). Differences between BSA-RhoB and corresponding NP were not statistically significant ( P .0.05). ( C ) Scheme of interaction of NP from monoclonal antibodies against HBsAg with the immobilized recombinant antigen. ( D ) ELISA data according to the scheme C. Abbreviations: HBSAg, hepatitis B virus S antigen; BSA, bovine serum albumin; NP, nanoparticles; Mab, monoclonal antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; o.u, optical units; RhoB, fluorescent rhodamine B dye; RFU, relative fluorescent units.
    Figure Legend Snippet: Results of binding of protein NP with specific ligands. Notes: ( A ) Scheme of interaction of BSA-RhoB NP with immobilized anti-BSA polyclonal antibodies. ( B ) Data of immunofluorescence assay according to scheme A. The error bars correspond to standard deviations (±SD). Differences between BSA-RhoB and corresponding NP were not statistically significant ( P .0.05). ( C ) Scheme of interaction of NP from monoclonal antibodies against HBsAg with the immobilized recombinant antigen. ( D ) ELISA data according to the scheme C. Abbreviations: HBSAg, hepatitis B virus S antigen; BSA, bovine serum albumin; NP, nanoparticles; Mab, monoclonal antibodies; HCV, hepatitis C virus; HBV, hepatitis B virus; o.u, optical units; RhoB, fluorescent rhodamine B dye; RFU, relative fluorescent units.

    Techniques Used: Binding Assay, Immunofluorescence, Recombinant, Enzyme-linked Immunosorbent Assay

    16) Product Images from "Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression"

    Article Title: Exploiting the Yeast L-A Viral Capsid for the In Vivo Assembly of Chimeric VLPs as Platform in Vaccine Development and Foreign Protein Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000415

    Sedimentation profile and electron microscopy of Gag/Δpp65 expressed in yeast demonstrate in vivo assembly into isometric VLP chimeras. (A) Western analysis of chimeric Gag/Δpp65 particles and natural L-A virions assembled in yeast and purified by ultracentrifugation through a linear sucrose gradient. Aliquots of each gradient fraction were separated by SDS-PAGE and probed with monoclonal anti-pp65 and polyclonal anti-Gag, respectively [GP, gradient pellet; M, full range rainbow marker, Amersham]. (B) Electron micrograph of sucrose-gradient-purified Gag/Δpp65 after negative staining with uranyl acetate/methyl cellulose [magnification 150,000; arrows indicate Gag/Δpp65 particles].
    Figure Legend Snippet: Sedimentation profile and electron microscopy of Gag/Δpp65 expressed in yeast demonstrate in vivo assembly into isometric VLP chimeras. (A) Western analysis of chimeric Gag/Δpp65 particles and natural L-A virions assembled in yeast and purified by ultracentrifugation through a linear sucrose gradient. Aliquots of each gradient fraction were separated by SDS-PAGE and probed with monoclonal anti-pp65 and polyclonal anti-Gag, respectively [GP, gradient pellet; M, full range rainbow marker, Amersham]. (B) Electron micrograph of sucrose-gradient-purified Gag/Δpp65 after negative staining with uranyl acetate/methyl cellulose [magnification 150,000; arrows indicate Gag/Δpp65 particles].

    Techniques Used: Sedimentation, Electron Microscopy, In Vivo, Western Blot, Purification, SDS Page, Marker, Negative Staining

    Chimeric Gag/K28α particles displaying the toxic α-subunit of the K28 virus toxin assemble into yeast VLPs that induce an in vivo antibody response in rabbit. (A) SDS-PAGE and Coomassie-Blue staining of recombinant Gag/K28α particles expressed and assembled in yeast and purified by sucrose gradient centrifugation. (B) Western analysis of the α/β heterodimeric K28 virus toxin probed with a rabbit polyclonal antiserum raised against chimeric Gag/K28α VLPs assembled in yeast. Positions of the heterodimeric K28 toxin (α/β) and its tetrameric derivative [α/β] 2 that forms spontaneously under conditions of a non-reducing SDS-PAGE are indicated (PI, pre-immune serum).
    Figure Legend Snippet: Chimeric Gag/K28α particles displaying the toxic α-subunit of the K28 virus toxin assemble into yeast VLPs that induce an in vivo antibody response in rabbit. (A) SDS-PAGE and Coomassie-Blue staining of recombinant Gag/K28α particles expressed and assembled in yeast and purified by sucrose gradient centrifugation. (B) Western analysis of the α/β heterodimeric K28 virus toxin probed with a rabbit polyclonal antiserum raised against chimeric Gag/K28α VLPs assembled in yeast. Positions of the heterodimeric K28 toxin (α/β) and its tetrameric derivative [α/β] 2 that forms spontaneously under conditions of a non-reducing SDS-PAGE are indicated (PI, pre-immune serum).

    Techniques Used: In Vivo, SDS Page, Staining, Recombinant, Purification, Gradient Centrifugation, Western Blot

    17) Product Images from "Conditional Ablation of the Neural Cell Adhesion Molecule Reduces Precision of Spatial Learning, Long-Term Potentiation, and Depression in the CA1 Subfield of Mouse Hippocampus"

    Article Title: Conditional Ablation of the Neural Cell Adhesion Molecule Reduces Precision of Spatial Learning, Long-Term Potentiation, and Depression in the CA1 Subfield of Mouse Hippocampus

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3298-03.2004

    Expression level of NCAM protein in brain subregions of NCAMff- and NCAMff+ mice. A , Western blot analyses were performed on tissue homogenates from olfactory bulb (ob), cerebral cortex (ctx), and hippocampus (hp) of mice homozygous for the NCAM-floxed allele and heterozygous for the αCaMKII-cre transgene (NCAMff+) and mice homozygous for the NCAM-floxed allele but negative for the αCaMKII-cre transgene (NCAMff-). Thirty micrograms of crude tissue lysates were loaded onto each lane. NCAM expression levels, as detected by polyclonal NCAM antibodies, were not different between the two genotypes in these brain regions at P9 (top panel), whereas at P112 (bottom panel), NCAM levels were decreased in the hippocampus of NCAMff+ mice. Protein loadings were controlled by GAPDH immunoreactivity. B , Western blot analysis of NCAM expression in the hippocampus of NCAMff- and NCAMff+ mice at P9, P15, P22, P49, and P112. Thirty micrograms of crude tissue lysates were loaded onto each lane. C , Eighty micrograms of crude tissue lysates of hippocampus isolated from 49-d-old NCAMff+ mutants (CKO) and a range of 0.7-50 μg of protein from hippocampus of 49-d-old NCAMff- controls (NCAM-floxed) were subjected to Western blot analysis using NCAM antibodies to estimate the efficiency of CaMKII-cre-mediated inactivation of NCAM expression in the hippocampus. All samples were pooled from two to three animals.
    Figure Legend Snippet: Expression level of NCAM protein in brain subregions of NCAMff- and NCAMff+ mice. A , Western blot analyses were performed on tissue homogenates from olfactory bulb (ob), cerebral cortex (ctx), and hippocampus (hp) of mice homozygous for the NCAM-floxed allele and heterozygous for the αCaMKII-cre transgene (NCAMff+) and mice homozygous for the NCAM-floxed allele but negative for the αCaMKII-cre transgene (NCAMff-). Thirty micrograms of crude tissue lysates were loaded onto each lane. NCAM expression levels, as detected by polyclonal NCAM antibodies, were not different between the two genotypes in these brain regions at P9 (top panel), whereas at P112 (bottom panel), NCAM levels were decreased in the hippocampus of NCAMff+ mice. Protein loadings were controlled by GAPDH immunoreactivity. B , Western blot analysis of NCAM expression in the hippocampus of NCAMff- and NCAMff+ mice at P9, P15, P22, P49, and P112. Thirty micrograms of crude tissue lysates were loaded onto each lane. C , Eighty micrograms of crude tissue lysates of hippocampus isolated from 49-d-old NCAMff+ mutants (CKO) and a range of 0.7-50 μg of protein from hippocampus of 49-d-old NCAMff- controls (NCAM-floxed) were subjected to Western blot analysis using NCAM antibodies to estimate the efficiency of CaMKII-cre-mediated inactivation of NCAM expression in the hippocampus. All samples were pooled from two to three animals.

    Techniques Used: Expressing, Mouse Assay, Western Blot, Isolation

    NCAM and PSA immunoreactivity in the brains of NCAMff- and NCAMff+ mice. A 1 - F 1 , Frontal brain sections of 63- to 70-d-old NCAMff- ( A 1 , D 1 ), NCAMff+ ( B 1 , E 1 ), and constitutive NCAM-deficient mice (NCAM-/-; C 1 , F 1 ) stained with polyclonal antibodies against all NCAM isoforms and with a monoclonal antibody to PSA. Note the normal expression pattern of NCAM and PSA in the cerebral cortex (cc), paraventricular thalamic nucleus (pvt), and hypothalamus (ht) in NCAMff- and NCAMff+ mice compared with constitutive NCAM-deficient (NCAM-/-) mice. Scale bar, 2.5 mm. A 2 - F 2 , NCAM and PSA immunoreactivity in the hippocampi of NCAMff-, NCAMff+, and NCAM-/- mice, corresponding to images shown in panels A 1 - F 1 . Loss of NCAM and PSA immunoreactivity in the molecular layer of the dentate gyrus (ml) and the mossy fibers (mf) is particularly obvious. Residual levels of NCAM and PSA remain in the inner granule cell layer (g), hilus (h), stratum lacunosum-moleculare (lm), and alveus (a). Scale bar, 0.5 mm.
    Figure Legend Snippet: NCAM and PSA immunoreactivity in the brains of NCAMff- and NCAMff+ mice. A 1 - F 1 , Frontal brain sections of 63- to 70-d-old NCAMff- ( A 1 , D 1 ), NCAMff+ ( B 1 , E 1 ), and constitutive NCAM-deficient mice (NCAM-/-; C 1 , F 1 ) stained with polyclonal antibodies against all NCAM isoforms and with a monoclonal antibody to PSA. Note the normal expression pattern of NCAM and PSA in the cerebral cortex (cc), paraventricular thalamic nucleus (pvt), and hypothalamus (ht) in NCAMff- and NCAMff+ mice compared with constitutive NCAM-deficient (NCAM-/-) mice. Scale bar, 2.5 mm. A 2 - F 2 , NCAM and PSA immunoreactivity in the hippocampi of NCAMff-, NCAMff+, and NCAM-/- mice, corresponding to images shown in panels A 1 - F 1 . Loss of NCAM and PSA immunoreactivity in the molecular layer of the dentate gyrus (ml) and the mossy fibers (mf) is particularly obvious. Residual levels of NCAM and PSA remain in the inner granule cell layer (g), hilus (h), stratum lacunosum-moleculare (lm), and alveus (a). Scale bar, 0.5 mm.

    Techniques Used: Mouse Assay, Staining, Expressing

    18) Product Images from "US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells"

    Article Title: US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.02151-18

    Cytoplasmic localization of VP8 at a late stage of BoHV-1 infection requires US3. (A to C) MDBK cells were infected with the indicated viruses at an MOI of 5 or mock-infected. Cells were processed for immunofluorescence at 4 and 8 hpi. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (D) Virus-infected MDBK cell lysates were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively.
    Figure Legend Snippet: Cytoplasmic localization of VP8 at a late stage of BoHV-1 infection requires US3. (A to C) MDBK cells were infected with the indicated viruses at an MOI of 5 or mock-infected. Cells were processed for immunofluorescence at 4 and 8 hpi. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (D) Virus-infected MDBK cell lysates were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively.

    Techniques Used: Infection, Immunofluorescence, Western Blot

    Phosphorylation by CK2 does not change the nuclear localization of VP8. (A) Mutating CK2-phosphorylated residues in VP8 does not affect the nuclear localization of VP8. EBTr cells were transfected with pFLAG-VP8 or pFLAG-VP8-M65-107 and fixed for immunofluorescent staining. (B) Overexpression of CK2α does not alter the VP8 nuclear localization. Plasmid pFLAG-VP8 or pFLAG-VP8-M65-107 was cotransfected with pCK2α-HA or individually transfected into COS-7 cells. The cells were fixed for immunofluorescent staining. VP8 was identified with polyclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. CK2α-HA was identified with monoclonal anti-HA antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.
    Figure Legend Snippet: Phosphorylation by CK2 does not change the nuclear localization of VP8. (A) Mutating CK2-phosphorylated residues in VP8 does not affect the nuclear localization of VP8. EBTr cells were transfected with pFLAG-VP8 or pFLAG-VP8-M65-107 and fixed for immunofluorescent staining. (B) Overexpression of CK2α does not alter the VP8 nuclear localization. Plasmid pFLAG-VP8 or pFLAG-VP8-M65-107 was cotransfected with pCK2α-HA or individually transfected into COS-7 cells. The cells were fixed for immunofluorescent staining. VP8 was identified with polyclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. CK2α-HA was identified with monoclonal anti-HA antibody followed by Alexa Fluor 633-conjugated goat anti-mouse IgG. The cell nuclei were identified with DAPI.

    Techniques Used: Transfection, Staining, Over Expression, Plasmid Preparation

    Nuclear lipid droplets are inversely correlated with phosphorylation of VP8 in infected cells. MDBK cells were infected with Wt BoHV-1, BoHV-1-YmVP8, or ΔUS3-BoHV-1 and fixed for immunostaining. VP8 (WT) and Mut-VP8 were detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.
    Figure Legend Snippet: Nuclear lipid droplets are inversely correlated with phosphorylation of VP8 in infected cells. MDBK cells were infected with Wt BoHV-1, BoHV-1-YmVP8, or ΔUS3-BoHV-1 and fixed for immunostaining. VP8 (WT) and Mut-VP8 were detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.

    Techniques Used: Infection, Immunostaining, Incubation

    US3-mediated phosphorylation promotes the cytoplasmic localization of VP8. (A) EBTr cells were transfected with different (combinations of) plasmids. Cells in panel 1 were transfected with pFLAG-VP8. Cells in panel 2 were cotransfected with pFLAG-VP8 and pUS3-HA. Cells in panel 3 were transfected with pFLAG-VP8-S16A. Cells in panel 4 were cotransfected with pFLAG-VP8-S16A and pUS3-HA. VP8 was identified with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (B) Relative quantification of cytoplasmic and nuclear VP8. The software Leica LAS AF Lite was used to analyze confocal pictures. The intensity of VP8 is represented as the fluorescence pixels at a wavelength of 488 nm. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: **, P ≤ 0.01. (C) The nuclear and cytoplasmic fractions were extracted from EBTr cells transfected with the indicated plasmids. VP8 and US3 in the nuclear and cytoplasmic fractions were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively. Nucleolin and tubulin were used to demonstrate the purity of nuclear and cytoplasmic fractions.
    Figure Legend Snippet: US3-mediated phosphorylation promotes the cytoplasmic localization of VP8. (A) EBTr cells were transfected with different (combinations of) plasmids. Cells in panel 1 were transfected with pFLAG-VP8. Cells in panel 2 were cotransfected with pFLAG-VP8 and pUS3-HA. Cells in panel 3 were transfected with pFLAG-VP8-S16A. Cells in panel 4 were cotransfected with pFLAG-VP8-S16A and pUS3-HA. VP8 was identified with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG, and US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The cell nuclei were identified with DAPI. (B) Relative quantification of cytoplasmic and nuclear VP8. The software Leica LAS AF Lite was used to analyze confocal pictures. The intensity of VP8 is represented as the fluorescence pixels at a wavelength of 488 nm. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: **, P ≤ 0.01. (C) The nuclear and cytoplasmic fractions were extracted from EBTr cells transfected with the indicated plasmids. VP8 and US3 in the nuclear and cytoplasmic fractions were analyzed by Western blotting. VP8 and US3 were detected with mouse anti-VP8 and rabbit anti-US3 antibodies followed by IRDye 800CW goat anti-mouse IgG and IRDye 680RD goat anti-rabbit IgG, respectively. Nucleolin and tubulin were used to demonstrate the purity of nuclear and cytoplasmic fractions.

    Techniques Used: Transfection, Software, Fluorescence, Western Blot

    VP8 is not colocalized with TGN protein AP1G1. (A) GOLGB1 is dispersed by BFA treatment. MDBK cells were treated with BFA. AP1G1 was detected with AP1G1-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. GOLGB1 was detected with GOLGB1-specific polyclonal antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. (B) VP8 is dispersed by BFA treatment. BoHV-1-infected MDBK cells were treated with BFA. VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.
    Figure Legend Snippet: VP8 is not colocalized with TGN protein AP1G1. (A) GOLGB1 is dispersed by BFA treatment. MDBK cells were treated with BFA. AP1G1 was detected with AP1G1-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. GOLGB1 was detected with GOLGB1-specific polyclonal antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. (B) VP8 is dispersed by BFA treatment. BoHV-1-infected MDBK cells were treated with BFA. VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG.

    Techniques Used: Infection

    VP8 causes formation of nuclear lipid droplets in transiently transfected cells. COS-7 cells transfected with different plasmids and Huh-7 cells were fixed for immunostaining. VP8 (WT) was detected with VP8-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Mut-VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.
    Figure Legend Snippet: VP8 causes formation of nuclear lipid droplets in transiently transfected cells. COS-7 cells transfected with different plasmids and Huh-7 cells were fixed for immunostaining. VP8 (WT) was detected with VP8-specific monoclonal antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was detected with US3-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Mut-VP8 was detected with VP8-specific polyclonal antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG. Cells were then incubated with Nile red and DAPI.

    Techniques Used: Transfection, Immunostaining, Incubation

    The amount of cytoplasmic FLAG-VP8 increases with the expression level of US3-HA. (A) The intensities of cytoplasmic and nuclear VP8. (B) The intensity of US3 in the nucleus. EBTr cells were transfected with pFLAG-VP8 and incubated for 12 h. This allowed VP8 to be expressed and localized to the nucleus. Subsequently, cells were transfected with pUS3-HA at 12 hpi, 17 hpi, or 22 hpi after transfection with pFLAG-VP8. All samples were fixed at 27 h for immunofluorescent staining, and the cell images were analyzed with the software Leica Application Suite X for protein quantification. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The intensities of fluorescence pixels at wavelengths of 488 nm and 633 nm were captured to indicate the quantity of VP8 and US3, respectively. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: *, 0.01
    Figure Legend Snippet: The amount of cytoplasmic FLAG-VP8 increases with the expression level of US3-HA. (A) The intensities of cytoplasmic and nuclear VP8. (B) The intensity of US3 in the nucleus. EBTr cells were transfected with pFLAG-VP8 and incubated for 12 h. This allowed VP8 to be expressed and localized to the nucleus. Subsequently, cells were transfected with pUS3-HA at 12 hpi, 17 hpi, or 22 hpi after transfection with pFLAG-VP8. All samples were fixed at 27 h for immunofluorescent staining, and the cell images were analyzed with the software Leica Application Suite X for protein quantification. VP8 was detected with monoclonal anti-VP8 antibody followed by Alexa Fluor 488-conjugated goat anti-mouse IgG. US3 was identified with polyclonal anti-US3 antibody followed by Alexa Fluor 633-conjugated goat anti-rabbit IgG. The intensities of fluorescence pixels at wavelengths of 488 nm and 633 nm were captured to indicate the quantity of VP8 and US3, respectively. The mean PDU are shown in the bar graphs. Error bars represent the SDs. The statistical significance is shown as follows: *, 0.01

    Techniques Used: Expressing, Transfection, Incubation, Staining, Software, Fluorescence

    19) Product Images from "Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum"

    Article Title: Transgenic expression of lactoferrin imparts enhanced resistance to head blight of wheat caused by Fusarium graminearum

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-33

    Expression of lactoferrin in seven T 8 transgenic wheat lines determined by Northern blot (A) and Western blot (B) analyses using two top leaves and inflorescences at growth stage Feekes 10.5 . Northern blot: Total RNA from transgenic and control plants were hybridized with a 32 P-labeled lactoferrin cDNA probe. 18s RNA was used as a loading control. Western blot: Immunodetection of lactoferrin protein in transgenic wheat plants using a polyclonal antibody reagent. Lane PC, purified lactoferrin protein; lane C, control wheat plant, lane 1-7 transgenic wheat lines: BLFW 119, 351, 378, 424, 685, 892 and 1102. Position of 85 kDa molecular weight marker is shown with an arrow on the right.
    Figure Legend Snippet: Expression of lactoferrin in seven T 8 transgenic wheat lines determined by Northern blot (A) and Western blot (B) analyses using two top leaves and inflorescences at growth stage Feekes 10.5 . Northern blot: Total RNA from transgenic and control plants were hybridized with a 32 P-labeled lactoferrin cDNA probe. 18s RNA was used as a loading control. Western blot: Immunodetection of lactoferrin protein in transgenic wheat plants using a polyclonal antibody reagent. Lane PC, purified lactoferrin protein; lane C, control wheat plant, lane 1-7 transgenic wheat lines: BLFW 119, 351, 378, 424, 685, 892 and 1102. Position of 85 kDa molecular weight marker is shown with an arrow on the right.

    Techniques Used: Expressing, Transgenic Assay, Northern Blot, Western Blot, Labeling, Immunodetection, Purification, Molecular Weight, Marker

    20) Product Images from "Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein"

    Article Title: Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein

    Journal: Molecular Biology of the Cell

    doi:

    (A) GRP125 from Dictyostelium and its closely related homologue. Western blot analysis of a lysate from vegetative D. discoideum cells using (1) the polyclonal anti-rabbit Dictyostelium GRP125/S3S4 antibody or (2) the polyclonal anti-rabbit Dictyostelium GRP125/S2S3 antibody. M, molecular weight marker. The anti-GRP125/S2S3 antibody monospecifically binds GRP125; the anti-GRP125/S3S4 antibody recognizes a second 125-kDa gelsolin-related protein. (B) Localization of GRP125 and its homologue to Triton X-100–soluble and –insoluble cellular fractions of Dictyostelium and extractability upon Ca 2+ treatment. Western blot analysis of subcellular fractions from D. discoideum after starvation in suspension using the anti-GRP125/S3S4 antibody. Shown are relative amounts of GRP125 and its homologue present in the Triton X-100–soluble cellular material, the Triton X-100–insoluble material extracted by Ca 2+ , and the detergent- and Ca 2+ -insoluble material. The fraction of GRP125 present in the Triton X-100–insoluble material is completely extracted by addition of Ca 2+ ; the GRP125 homologue is not solubilized by Ca 2+ . (C) Presence of GRP125-related proteins in cells from lower to higher eukaryotes. Western blot analysis of cell extracts from different sources using the anti-GRP125/S3S4 antibody. M, molecular weight marker. Next to 1 ( D. discoideum AX2 [control]), cell lysates were tested from Saccharomyces cerevisiae (2), S. pombe (3), human hepatocytes (4), Cv-1 cells (5), SF9 cells (6), mouse brain tissue (7), macrophages (8), muscle tissue of mouse (9), rat (10), and human (11), and NIH/3T3 fibroblasts (12). The polyclonal antibody reacted specifically with distinct proteins of differing sizes in a range of cell types. Prominent bands marked in the lysates are highlighted by arrows; arrow numbers code for the cell source.
    Figure Legend Snippet: (A) GRP125 from Dictyostelium and its closely related homologue. Western blot analysis of a lysate from vegetative D. discoideum cells using (1) the polyclonal anti-rabbit Dictyostelium GRP125/S3S4 antibody or (2) the polyclonal anti-rabbit Dictyostelium GRP125/S2S3 antibody. M, molecular weight marker. The anti-GRP125/S2S3 antibody monospecifically binds GRP125; the anti-GRP125/S3S4 antibody recognizes a second 125-kDa gelsolin-related protein. (B) Localization of GRP125 and its homologue to Triton X-100–soluble and –insoluble cellular fractions of Dictyostelium and extractability upon Ca 2+ treatment. Western blot analysis of subcellular fractions from D. discoideum after starvation in suspension using the anti-GRP125/S3S4 antibody. Shown are relative amounts of GRP125 and its homologue present in the Triton X-100–soluble cellular material, the Triton X-100–insoluble material extracted by Ca 2+ , and the detergent- and Ca 2+ -insoluble material. The fraction of GRP125 present in the Triton X-100–insoluble material is completely extracted by addition of Ca 2+ ; the GRP125 homologue is not solubilized by Ca 2+ . (C) Presence of GRP125-related proteins in cells from lower to higher eukaryotes. Western blot analysis of cell extracts from different sources using the anti-GRP125/S3S4 antibody. M, molecular weight marker. Next to 1 ( D. discoideum AX2 [control]), cell lysates were tested from Saccharomyces cerevisiae (2), S. pombe (3), human hepatocytes (4), Cv-1 cells (5), SF9 cells (6), mouse brain tissue (7), macrophages (8), muscle tissue of mouse (9), rat (10), and human (11), and NIH/3T3 fibroblasts (12). The polyclonal antibody reacted specifically with distinct proteins of differing sizes in a range of cell types. Prominent bands marked in the lysates are highlighted by arrows; arrow numbers code for the cell source.

    Techniques Used: Western Blot, Molecular Weight, Marker

    (A) Schematic presentation of the replacement strategy yielding the inactivation of the GRP125 gene in Dictyostelium . The 1.8-kb genomic region at the 5′ end of the GRP125 gene was amplified via PCR (small arrows) and cloned into pGEM-5Z. The Eco RI– Eco RI-fragment (light gray) was deleted (flash in light gray) (1) and replaced by blunt-end insertion of the Bsr resistance cassette (2), Act15P, actin 15 promotor; Act8T, actin 8 terminator; Bsr, blasticidin-S deaminase gene. Restriction sites marked in gray got lost (cross) or introduced in the replacement construct. A kilobase ruler refers to nucleotide positions in the GRP125 gene (A of ATG = 1). (B) Western blot analysis of homogenates of three independent GRP125-deficient mutants (GRP125 − /17, GRP125 − /18, and GRP125 − /C)and AX2 cells. Cell lysates (1 × 10 6 cells, growth phase) were analyzed with the polyclonal anti-GRP125/S3S4 antibody. GRP125 − /17, GRP125 − /18, and GRP125 − /C fail to express GRP125 but produce the GRP125 homologue. (C) Comparative Southern blot analysis of genomic DNA of GRP125-deficient cells (GRP125 − /17 [1], GRP125 − /18 [2], and GRP125 − /C [3]) and AX2 cells (4). Genomic DNA from D. discoideum AX2 and the mutants was Eco RI– Bgl II double digested, size fractionated by electrophoresis in agarose gels, and transferred to a Hybond-N membrane. The blot was hybridized to radioactively labeled DNA probes of GRP125 corresponding to a fragment exactly 3′ of the deleted Eco RI– Eco RI fragment (GRP125-specific probe F1, bp 1228–2727 of the GRP125 coding region) or the Bsr encoding sequence.
    Figure Legend Snippet: (A) Schematic presentation of the replacement strategy yielding the inactivation of the GRP125 gene in Dictyostelium . The 1.8-kb genomic region at the 5′ end of the GRP125 gene was amplified via PCR (small arrows) and cloned into pGEM-5Z. The Eco RI– Eco RI-fragment (light gray) was deleted (flash in light gray) (1) and replaced by blunt-end insertion of the Bsr resistance cassette (2), Act15P, actin 15 promotor; Act8T, actin 8 terminator; Bsr, blasticidin-S deaminase gene. Restriction sites marked in gray got lost (cross) or introduced in the replacement construct. A kilobase ruler refers to nucleotide positions in the GRP125 gene (A of ATG = 1). (B) Western blot analysis of homogenates of three independent GRP125-deficient mutants (GRP125 − /17, GRP125 − /18, and GRP125 − /C)and AX2 cells. Cell lysates (1 × 10 6 cells, growth phase) were analyzed with the polyclonal anti-GRP125/S3S4 antibody. GRP125 − /17, GRP125 − /18, and GRP125 − /C fail to express GRP125 but produce the GRP125 homologue. (C) Comparative Southern blot analysis of genomic DNA of GRP125-deficient cells (GRP125 − /17 [1], GRP125 − /18 [2], and GRP125 − /C [3]) and AX2 cells (4). Genomic DNA from D. discoideum AX2 and the mutants was Eco RI– Bgl II double digested, size fractionated by electrophoresis in agarose gels, and transferred to a Hybond-N membrane. The blot was hybridized to radioactively labeled DNA probes of GRP125 corresponding to a fragment exactly 3′ of the deleted Eco RI– Eco RI fragment (GRP125-specific probe F1, bp 1228–2727 of the GRP125 coding region) or the Bsr encoding sequence.

    Techniques Used: Amplification, Polymerase Chain Reaction, Clone Assay, Construct, Western Blot, Southern Blot, Electrophoresis, Labeling, Sequencing

    (A–K) GRP125 and its homologue localize to vesicular compartments in Dictyostelium cells. Corresponding phase-contrast micrographs (A, C, E, G, and I) and fluorescence images (B, D, F, H, and K) at different developmental stages are shown. Bar, 10 μm. (A–D) Localization of GRP125 in growth phase (A and B) and aggregation-competent (C and D) D. discoideum AX2 cells. Fixation was achieved using cold methanol; specimens were processed for indirect immunofluorescence labeling of GRP125 using the polyclonal anti-GRP125/S2S3 antibody followed by Cy3-labeled goat anti-rabbit IgG. (E–H) Immunofluorescence localization of both GRP125 and its homologue in growth phase (E and F) and aggregation-competent (G and H) Dictyostelium cells. Cells were fixed with picric acid/formaldehyde/70% ethanol. Labeling of both GRP125 and its homologue was performed using the polyclonal anti-GRP125/S3S4 antibody followed by the Cy3-labeled goat-anti rabbit IgG. (I and K) Background fluorescence using only the secondary Cy3 labeled goat-anti rabbit IgG antibody. (L and M) Incorporation of the recombinant rhodamine-labeled GRP125/S3S4 polypeptide to vesicle-like subcellular compartments after microinjection into living NIH/3T3 fibroblasts. (M) Fluorescence; (L) corresponding phase-contrast image. Bar, 10 μm.
    Figure Legend Snippet: (A–K) GRP125 and its homologue localize to vesicular compartments in Dictyostelium cells. Corresponding phase-contrast micrographs (A, C, E, G, and I) and fluorescence images (B, D, F, H, and K) at different developmental stages are shown. Bar, 10 μm. (A–D) Localization of GRP125 in growth phase (A and B) and aggregation-competent (C and D) D. discoideum AX2 cells. Fixation was achieved using cold methanol; specimens were processed for indirect immunofluorescence labeling of GRP125 using the polyclonal anti-GRP125/S2S3 antibody followed by Cy3-labeled goat anti-rabbit IgG. (E–H) Immunofluorescence localization of both GRP125 and its homologue in growth phase (E and F) and aggregation-competent (G and H) Dictyostelium cells. Cells were fixed with picric acid/formaldehyde/70% ethanol. Labeling of both GRP125 and its homologue was performed using the polyclonal anti-GRP125/S3S4 antibody followed by the Cy3-labeled goat-anti rabbit IgG. (I and K) Background fluorescence using only the secondary Cy3 labeled goat-anti rabbit IgG antibody. (L and M) Incorporation of the recombinant rhodamine-labeled GRP125/S3S4 polypeptide to vesicle-like subcellular compartments after microinjection into living NIH/3T3 fibroblasts. (M) Fluorescence; (L) corresponding phase-contrast image. Bar, 10 μm.

    Techniques Used: Fluorescence, Immunofluorescence, Labeling, Recombinant

    21) Product Images from "Myr 8, A Novel Unconventional Myosin Expressed during Brain Development Associates with the Protein Phosphatase Catalytic Subunits 1α and 1γ1"

    Article Title: Myr 8, A Novel Unconventional Myosin Expressed during Brain Development Associates with the Protein Phosphatase Catalytic Subunits 1α and 1γ1

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.21-20-07954.2001

    Myr 8b associates with the protein phosphatase catalytic subunits 1α and 1γ1. A crude cytoskeletal/membrane fraction prepared from postnatal day 10 cerebellum was solubilized in 1% Triton X-100, and unsolubilized components were sedimented by centrifugation. Aliquots of the detergent lysate were processed for immunoprecipitation using affinity-purified antibodies directed toward the C-terminal tail domain of myr 8b. Immunoprecipitated polypeptides were resolved by SDS-PAGE and processed for immunoblot analyses using polyclonal antiserum directed against multiple (1α, 1β, 1γ, 2A, 2B, and X) and individual ( PP1α ), ( PP1 β), ( PP1 γ), and ( PP2A ) protein phosphatase catalytic subunits and actin. Molecular mass × 10 − 3 is indicated vertically .
    Figure Legend Snippet: Myr 8b associates with the protein phosphatase catalytic subunits 1α and 1γ1. A crude cytoskeletal/membrane fraction prepared from postnatal day 10 cerebellum was solubilized in 1% Triton X-100, and unsolubilized components were sedimented by centrifugation. Aliquots of the detergent lysate were processed for immunoprecipitation using affinity-purified antibodies directed toward the C-terminal tail domain of myr 8b. Immunoprecipitated polypeptides were resolved by SDS-PAGE and processed for immunoblot analyses using polyclonal antiserum directed against multiple (1α, 1β, 1γ, 2A, 2B, and X) and individual ( PP1α ), ( PP1 β), ( PP1 γ), and ( PP2A ) protein phosphatase catalytic subunits and actin. Molecular mass × 10 − 3 is indicated vertically .

    Techniques Used: Centrifugation, Immunoprecipitation, Affinity Purification, SDS Page

    22) Product Images from "Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response"

    Article Title: Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response

    Journal: Journal of Inflammation (London, England)

    doi: 10.1186/1476-9255-2-16

    Chemokine secretion by stimulated primary small airway epithelial cells (SAEC) . (A) SAEC were stimulated for 24 h with different TLR ligands including poly(I:C), flagellin, zymosan and macrophage activating lipopetide-2 (MALP-2) and cell supernatants were analyzed using multiplex ELISAs. Results are shown as fold changes relative to untreated controls. The figure shows representative results of three independent experiments. Stimulation of the cells with LPS or PGN had no significant effect on the secretion of the indicated chemokines (data not shown). (B) Inhibition of poly(I:C) induced IP-10 secretion by a monoclonal, functional blocking anti-TLR3 antibody. SAEC were stimulated in the presence of an anti-TLR3 antibody or an IgG 1 isotype control with 5 μg/ml poly(I:C) for 6 h and IP-10 secretion was analyzed in triplicates using an IP-10 ELISA. Results were compared to untreated controls and poly(I:C) stimulated cells in the absence of an antibody. (C) IFN-β secretion of SAEC after stimulation with poly(I:C). SAEC were stimulated with 5 μg/ml poly(I:C) for 6 h or 24 h and IFN-β secretion was analyzed in triplicates using an IFN-β ELISA. Results were compared to untreated controls. (D) Inhibition of poly(I:C) induced IP-10 and I-TAC expression by a goat polyclonal functional blocking anti-IFN-β antibody. SAEC were stimulated in the presence of a goat anti-IFN-β antibody (1 or 5 μg/ml; gray bars) or a goat IgG control (1 or 5 μg/ml; white bars) with 5 μg/ml poly(I:C) for 6 h. IP-10 or I-TAC expression was analyzed by real-time RT-PCR. Expression data were normalized using β-actin as endogenous control and are shown as fold changes relative to untreated controls. Results were compared to poly(I:C) stimulated cells in the absence of an antibody (black bars).
    Figure Legend Snippet: Chemokine secretion by stimulated primary small airway epithelial cells (SAEC) . (A) SAEC were stimulated for 24 h with different TLR ligands including poly(I:C), flagellin, zymosan and macrophage activating lipopetide-2 (MALP-2) and cell supernatants were analyzed using multiplex ELISAs. Results are shown as fold changes relative to untreated controls. The figure shows representative results of three independent experiments. Stimulation of the cells with LPS or PGN had no significant effect on the secretion of the indicated chemokines (data not shown). (B) Inhibition of poly(I:C) induced IP-10 secretion by a monoclonal, functional blocking anti-TLR3 antibody. SAEC were stimulated in the presence of an anti-TLR3 antibody or an IgG 1 isotype control with 5 μg/ml poly(I:C) for 6 h and IP-10 secretion was analyzed in triplicates using an IP-10 ELISA. Results were compared to untreated controls and poly(I:C) stimulated cells in the absence of an antibody. (C) IFN-β secretion of SAEC after stimulation with poly(I:C). SAEC were stimulated with 5 μg/ml poly(I:C) for 6 h or 24 h and IFN-β secretion was analyzed in triplicates using an IFN-β ELISA. Results were compared to untreated controls. (D) Inhibition of poly(I:C) induced IP-10 and I-TAC expression by a goat polyclonal functional blocking anti-IFN-β antibody. SAEC were stimulated in the presence of a goat anti-IFN-β antibody (1 or 5 μg/ml; gray bars) or a goat IgG control (1 or 5 μg/ml; white bars) with 5 μg/ml poly(I:C) for 6 h. IP-10 or I-TAC expression was analyzed by real-time RT-PCR. Expression data were normalized using β-actin as endogenous control and are shown as fold changes relative to untreated controls. Results were compared to poly(I:C) stimulated cells in the absence of an antibody (black bars).

    Techniques Used: Multiplex Assay, Inhibition, Functional Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

    23) Product Images from "The TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution"

    Article Title: The TetL tetracycline efflux protein from Bacillus subtilis is a dimer in the membrane and in detergent solution

    Journal: Biochemistry

    doi: 10.1021/bi035173q

    Expression and purification of TetL. A. Silver-stained SDS-PAGE. B. Western blot analysis using polyclonal anti-TetL antibodies. C. Western blot analysis using India His-Probe-HRP. Dimers are detected in purified TetL samples by both Coomassie-blue stained SDS-PAGE and Western blot. Low molecular weight fragments of TetL and high-molecular weight aggregates can be seen in B. They did not co-purify with intact transporter protein. Four samples were shown in each panel: solubilized membrane, Co 2+ -TALON-purified sample, thrombin-digested sample and SE-column purified sample.
    Figure Legend Snippet: Expression and purification of TetL. A. Silver-stained SDS-PAGE. B. Western blot analysis using polyclonal anti-TetL antibodies. C. Western blot analysis using India His-Probe-HRP. Dimers are detected in purified TetL samples by both Coomassie-blue stained SDS-PAGE and Western blot. Low molecular weight fragments of TetL and high-molecular weight aggregates can be seen in B. They did not co-purify with intact transporter protein. Four samples were shown in each panel: solubilized membrane, Co 2+ -TALON-purified sample, thrombin-digested sample and SE-column purified sample.

    Techniques Used: Expressing, Purification, Staining, SDS Page, Western Blot, Molecular Weight

    24) Product Images from "Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model"

    Article Title: Expression of verocytotoxic Escherichia coli antigens in tobacco seeds and evaluation of gut immunity after oral administration in mouse model

    Journal: Journal of Veterinary Science

    doi: 10.4142/jvs.2013.14.3.263

    Agglutination on slides with F18+ polyclonal serum. (A) Total protein extracted from wild-type seeds. (B) Total protein extracted from F18+ seeds.
    Figure Legend Snippet: Agglutination on slides with F18+ polyclonal serum. (A) Total protein extracted from wild-type seeds. (B) Total protein extracted from F18+ seeds.

    Techniques Used: Agglutination

    25) Product Images from "Cellular and molecular cues of glucose sensing in the rat olfactory bulb"

    Article Title: Cellular and molecular cues of glucose sensing in the rat olfactory bulb

    Journal: Frontiers in Neuroscience

    doi: 10.3389/fnins.2014.00333

    GLUT4 localization in the OB layers . Representative GLUT4 immunostaining of the main OB layers of adult rats performed with monoclonal (A) , and polyclonal antibodies (B–H) merged with the nuclear marker DAPI (A–C) , double immunostained with IR (D–F) , with MAP2, a dendritic processes marker (G) and with Synapsin 1, a presynaptic marker (H) . (A–C) In the glomerular layer, glomeruli show different staining intensities (strongly labeled: #, slightly labeled: §; not labeled: * ). Scattered periglomerular cells ( A ; arrows) around unstained glomeruli ( * ) are GLUT4 positive. Some mitral cells ( A ; arrowhead) are intensively immunostained. (C) Within glomeruli the GLUT4 immunostaining is heterogeneous with alternation of highly marked zones and faded zones (delimited by dotted lines). (D) In the mitral cell layer, scattered mitral cells co-express GLUT4 and IR (arrows) while others express only IR (arrowhead). (E,F) Within glomeruli expressing GLUT4, at high magnification in satiated rats (E) , GLUT4 was found enclosing IR clusters located within large processes ( E ; arrows). In fasted rats (F) , GLUT4 was either co-localized ( F : small arrowhead) or not (arrow) in the cytoplasm of these processes. Some processes expressed only IR (large arrowhead). (G,H) Immunofluorescence Apotome images of GLUT4, MAP2 and Synapsin 1 within glomeruli. Projection images of consecutive x-y optical sections are shown in the upper large panels. An x-z vertical scanning image of each upper panel is shown in the lower small panel. Green and blue signals indicate GLUT4 and nuclei (DAPI) respectively, and orange indicates MAP2 (G) and Synapsin 1 (H) . MAP2 can be co-localized with GLUT4 ( G , arrows in lower small panel; yellow signal) while Synapsin 1 is located close to GLUT4 ( H , arrows in lower small panel). (I) Control section where primary antibodies were omitted: only the nuclear marker DAPI was detected. (NL, nerve layer; EPL, external plexiform layer; GL, glomerular layer; MCL, mitral cell layer; GCL, granular cell layer; MAP2, microtubule-associated protein 2; Syn, Synapsin 1; IR, insulin receptor; Sat., Satiated rat; Fast, Fasted rat).
    Figure Legend Snippet: GLUT4 localization in the OB layers . Representative GLUT4 immunostaining of the main OB layers of adult rats performed with monoclonal (A) , and polyclonal antibodies (B–H) merged with the nuclear marker DAPI (A–C) , double immunostained with IR (D–F) , with MAP2, a dendritic processes marker (G) and with Synapsin 1, a presynaptic marker (H) . (A–C) In the glomerular layer, glomeruli show different staining intensities (strongly labeled: #, slightly labeled: §; not labeled: * ). Scattered periglomerular cells ( A ; arrows) around unstained glomeruli ( * ) are GLUT4 positive. Some mitral cells ( A ; arrowhead) are intensively immunostained. (C) Within glomeruli the GLUT4 immunostaining is heterogeneous with alternation of highly marked zones and faded zones (delimited by dotted lines). (D) In the mitral cell layer, scattered mitral cells co-express GLUT4 and IR (arrows) while others express only IR (arrowhead). (E,F) Within glomeruli expressing GLUT4, at high magnification in satiated rats (E) , GLUT4 was found enclosing IR clusters located within large processes ( E ; arrows). In fasted rats (F) , GLUT4 was either co-localized ( F : small arrowhead) or not (arrow) in the cytoplasm of these processes. Some processes expressed only IR (large arrowhead). (G,H) Immunofluorescence Apotome images of GLUT4, MAP2 and Synapsin 1 within glomeruli. Projection images of consecutive x-y optical sections are shown in the upper large panels. An x-z vertical scanning image of each upper panel is shown in the lower small panel. Green and blue signals indicate GLUT4 and nuclei (DAPI) respectively, and orange indicates MAP2 (G) and Synapsin 1 (H) . MAP2 can be co-localized with GLUT4 ( G , arrows in lower small panel; yellow signal) while Synapsin 1 is located close to GLUT4 ( H , arrows in lower small panel). (I) Control section where primary antibodies were omitted: only the nuclear marker DAPI was detected. (NL, nerve layer; EPL, external plexiform layer; GL, glomerular layer; MCL, mitral cell layer; GCL, granular cell layer; MAP2, microtubule-associated protein 2; Syn, Synapsin 1; IR, insulin receptor; Sat., Satiated rat; Fast, Fasted rat).

    Techniques Used: Immunostaining, Marker, Staining, Labeling, Expressing, Immunofluorescence

    26) Product Images from "Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells"

    Article Title: Role of Integrins in the Assembly and Function of Hensin in Intercalated Cells

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2007070737

    Integrin expression in clone C cells. (A) Expression patterns of integrin β1, αv, α6, and α1 in confluent monolayers of clone C cells seeded at LD and HD. Integrin staining is depicted in red, and nuclear staining with Sytox is shown in green. Integrin α3 staining was also observed by immunostaining but not depicted here. (B) Integrin expression in clone C cells was investigated by basolateral biotinylation followed by immunoprecipitation with integrin antibodies as indicated and Western blotted with anti-streptavidin antibodies (for αv and α3 in the top panel and all lanes in the bottom panel). In addition, Western blots were performed on biotin-streptavidin–purified basolateral membranes from HD cells with integrin α1, α5, and α6. (C) Western blots of cell lysates from LD and HD cells using the polyclonal antibody directed against extracellular domain of rabbit integrin β1 (left). (Right) Western blot using the same antibody in the presence of 600 ng/ml recombinant peptide immunogen. (D) Flow cytometry analysis of clone C cells with various integrin antibodies as indicated in the labels.
    Figure Legend Snippet: Integrin expression in clone C cells. (A) Expression patterns of integrin β1, αv, α6, and α1 in confluent monolayers of clone C cells seeded at LD and HD. Integrin staining is depicted in red, and nuclear staining with Sytox is shown in green. Integrin α3 staining was also observed by immunostaining but not depicted here. (B) Integrin expression in clone C cells was investigated by basolateral biotinylation followed by immunoprecipitation with integrin antibodies as indicated and Western blotted with anti-streptavidin antibodies (for αv and α3 in the top panel and all lanes in the bottom panel). In addition, Western blots were performed on biotin-streptavidin–purified basolateral membranes from HD cells with integrin α1, α5, and α6. (C) Western blots of cell lysates from LD and HD cells using the polyclonal antibody directed against extracellular domain of rabbit integrin β1 (left). (Right) Western blot using the same antibody in the presence of 600 ng/ml recombinant peptide immunogen. (D) Flow cytometry analysis of clone C cells with various integrin antibodies as indicated in the labels.

    Techniques Used: Expressing, Staining, Immunostaining, Immunoprecipitation, Western Blot, Purification, Recombinant, Flow Cytometry, Cytometry

    27) Product Images from "Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein"

    Article Title: Anti-HSP90 autoantibodies in sera of infertile women identify a dominant, conserved epitope EP6 (380-389) of HSP90 beta protein

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/1477-7827-9-16

    Cellular characterization of epitope EP6 . Indirect immunofluorescence studies using the anti-EP6 peptide polyclonal antibodies. Immunoreactivity (green stain) as seen in panel (A) ovulated oocyte, (B) 2 cell embryo, (C) 4 cell embryo, (D) 8 cell embryo and (E) morula. The pre-immune of the same animal showed no immunostaining to any of the cell types as represented in bright field merged panel indicating the nucleus and cell boundary (F). DAPI (blue stain) was used to counterstain the nucleus. Images were taken on Carl Zeiss Confocal microscope at X640 magnification.
    Figure Legend Snippet: Cellular characterization of epitope EP6 . Indirect immunofluorescence studies using the anti-EP6 peptide polyclonal antibodies. Immunoreactivity (green stain) as seen in panel (A) ovulated oocyte, (B) 2 cell embryo, (C) 4 cell embryo, (D) 8 cell embryo and (E) morula. The pre-immune of the same animal showed no immunostaining to any of the cell types as represented in bright field merged panel indicating the nucleus and cell boundary (F). DAPI (blue stain) was used to counterstain the nucleus. Images were taken on Carl Zeiss Confocal microscope at X640 magnification.

    Techniques Used: Immunofluorescence, Staining, Immunostaining, Microscopy

    Biochemical characterization of epitope EP6 . Panel (A) depicts dot blot analysis using rabbit polyclonal antibodies to peptides EP6. EP6 in panel A6 shows strong immunoreactivity with the post-immune sera. The pre-immune of the same rabbit did not react to any of the 10 peptides EP1 to EP10 in sequential order. Panel (B): Western blot analysis using rabbit polyclonal antibodies to peptide EP6 with recombinant HSP90 loaded in duplicates. B1: Pre immune serum shows no immunoreactivity. B2: Post immune serum reacts with the recombinant protein. Panel (C): Western blot analysis using the anti-EP6 peptide polyclonal antibodies with crude mice ovarian protein. Lane RO: total mice ovarian extract stained with Coomassie blue stain with an arrow indicating the locus representing the HSP90 protein. Lane Pre: The pre immune serum shows no immunoreactivity. Lane Post: The post-immune sera shows single band reactivity in the lane marked at the 90 kDa. Lane NC: Secondary alone control shows no reactivity.
    Figure Legend Snippet: Biochemical characterization of epitope EP6 . Panel (A) depicts dot blot analysis using rabbit polyclonal antibodies to peptides EP6. EP6 in panel A6 shows strong immunoreactivity with the post-immune sera. The pre-immune of the same rabbit did not react to any of the 10 peptides EP1 to EP10 in sequential order. Panel (B): Western blot analysis using rabbit polyclonal antibodies to peptide EP6 with recombinant HSP90 loaded in duplicates. B1: Pre immune serum shows no immunoreactivity. B2: Post immune serum reacts with the recombinant protein. Panel (C): Western blot analysis using the anti-EP6 peptide polyclonal antibodies with crude mice ovarian protein. Lane RO: total mice ovarian extract stained with Coomassie blue stain with an arrow indicating the locus representing the HSP90 protein. Lane Pre: The pre immune serum shows no immunoreactivity. Lane Post: The post-immune sera shows single band reactivity in the lane marked at the 90 kDa. Lane NC: Secondary alone control shows no reactivity.

    Techniques Used: Dot Blot, Western Blot, Recombinant, Mouse Assay, Staining

    28) Product Images from "Regulation of microtubule-based transport by MAP4"

    Article Title: Regulation of microtubule-based transport by MAP4

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E14-01-0022

    XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150 Glued subunit of the dynactin complex (p150 Glued ); p150 Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150 Glued ; p150 Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.
    Figure Legend Snippet: XMAP4 binds dynactin but not MT motor proteins. (A) Whole-cell extracts or immunoprecipitates of EGFP-XMAP4 with rabbit polyclonal antibodies against EGFP probed with mouse monoclonal antibodies specific for EGFP (EGFP), Kif3A subunit of kinesin-2 (Kif3A), dynein IC (DIC), or the p150 Glued subunit of the dynactin complex (p150 Glued ); p150 Glued but not kinesin-2 or dynein coimmunoprecipitates with EGFP-XMAP4. (B) Immunoprecipitate of the endogenous XMAP4 with antibodies against XMAP4 MBD probed with an antibody against p150 Glued ; p150 Glued coimmunoprecipitates with endogenous XMAP4. E, whole-cell extract; IP, immuno­precipitate.

    Techniques Used:

    29) Product Images from "Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function"

    Article Title: Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2007070770

    GFP expression in Pkhd1 mutant mice. (A) E15.5 Pkhd1 −/− kidney, liver, adrenal gland, and gastrointestinal tract stained with IHC using an anti-GFP polyclonal antibody. Positive signals were seen in the cortical adrenal cells (left arrow), periportal liver cells (right arrow), gastrointestinal tract (white box), and weakly positive staining was seen in the renal tubules (black box). (B) Higher magnification of the black box in A shows positive staining of the renal epithelia (arrow). (C) Higher magnification of the white box in A shows strong positive staining in the mucosal cells of the colon (arrow). (D) The differential interface contrast view showed GFP-positive staining (arrows) in the renal tubule epithelia of 4-mo-old Pkhd1 +/− (D, top) and Pkhd1 −/− (D, bottom) littermates. (E) Positive GFP staining was detected in an alveolar bronchiole of the lung in a 2-mo-old homozygous mouse (arrow). (F) Positive GFP was also seen in the tracheal epithelium of the same mouse (arrow). (G) GFP-positive staining (red) appeared in the ependymal cells lining the ventricles of the brain in a 2-mo-old homozygous mouse (arrow). Yo-pro (green) was used to stain the nuclei of cells. (H) GFP-positive staining (red) was observed in the diseased liver of a 2-mo-old homozygous mouse (arrows). (I) Cytokeratin 7–positive staining (green), a marker for epithelial cells, outlined the biliary epithelial structures (arrows). (J) The merged confocal image showed that GFP co-localized with cytokeratin 7. Bar = 30 μm in A; 15 μm in B and C; and 10 μm in D through J.
    Figure Legend Snippet: GFP expression in Pkhd1 mutant mice. (A) E15.5 Pkhd1 −/− kidney, liver, adrenal gland, and gastrointestinal tract stained with IHC using an anti-GFP polyclonal antibody. Positive signals were seen in the cortical adrenal cells (left arrow), periportal liver cells (right arrow), gastrointestinal tract (white box), and weakly positive staining was seen in the renal tubules (black box). (B) Higher magnification of the black box in A shows positive staining of the renal epithelia (arrow). (C) Higher magnification of the white box in A shows strong positive staining in the mucosal cells of the colon (arrow). (D) The differential interface contrast view showed GFP-positive staining (arrows) in the renal tubule epithelia of 4-mo-old Pkhd1 +/− (D, top) and Pkhd1 −/− (D, bottom) littermates. (E) Positive GFP staining was detected in an alveolar bronchiole of the lung in a 2-mo-old homozygous mouse (arrow). (F) Positive GFP was also seen in the tracheal epithelium of the same mouse (arrow). (G) GFP-positive staining (red) appeared in the ependymal cells lining the ventricles of the brain in a 2-mo-old homozygous mouse (arrow). Yo-pro (green) was used to stain the nuclei of cells. (H) GFP-positive staining (red) was observed in the diseased liver of a 2-mo-old homozygous mouse (arrows). (I) Cytokeratin 7–positive staining (green), a marker for epithelial cells, outlined the biliary epithelial structures (arrows). (J) The merged confocal image showed that GFP co-localized with cytokeratin 7. Bar = 30 μm in A; 15 μm in B and C; and 10 μm in D through J.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Staining, Immunohistochemistry, Marker

    Molecular relationship between FPC and PC2. (A) Using the anti-PC2 mAb hPKD2-Cm1A11, Western blot of duplicate protein lysates from WT, Pkhd1 +/− , and Pkhd1 −/− E13.5 littermates showed a significant downregulation of PC2 in Pkhd1 −/− embryos, indicating that lack of FPC inhibits PC2 expression in vivo . An anti–β-actin antibody was used for a protein-loading control. (B) In comparison with the WT littermate (left), IHC staining with the anti-PC2 polyclonal antibody hPKD2-Cp showed a significant decrease in PC2 expression in the cortical region of the 1-mo-old Pkhd1 −/− kidney (right). (C) Duplicate lysates from E13.5 WT, Pkhd1 +/− , and Pkhd1 −/− littermates were used to perform a co-IP Western using the anti-FPC antibody hAR-Nm3G12 to IP and the anti-PC2 antibody hPKD2-Cm1A11 to detect PC2 expression. Positive immunoreactivity was seen in the WT embryo, and progressively reduced immunoreactivities were seen in the Pkhd1 +/− and Pkhd1 −/− littermates, suggesting that FPC binds to PC2 in vivo . (D) Lysates from E13.5 WT, Pkd2 +/− , and Pkd2 −/− littermates were used to perform a co-IP Western using the anti-PC2 antibody to IP and the anti-FPC antibody to detect FPC expression. Positive immunoreactivity was seen in the WT and Pkd2 +/− littermates, but no immunoreactivity was observed in the Pkd2 −/− littermate, providing further evidence that FPC physically interacts with PC2 in vivo . (E) There was no change in FPC expression in Western blot analysis among the WT and Pkd2 −/− littermates, indicating that the downregulation of PC2 does not affect FPC expression. (F) HA- and Flag-tagged expression vectors, in which the COOH-terminus of FPC (FPC-C-Flag) and the NH2-terminus of PC2 (PC2-N-HA) were constructed in-frame, were transiently co-transfected into HEK293 cells. Using an anti-Flag antibody to IP and an anti-HA antibody to detect the NH2-terminus of PC2, positive immunoreactivity was seen only in the co-transfected sample, indicating that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2 in vitro . (G) The same FPC-C-Flag expression vector was transiently co-transfected into HEK293 cells with an expression vector containing the human full-length PKD2 cDNA (PC2-Full). The anti-PC2 antibody hPKD2-Cm1A11 was used for IP and an anti-Flag antibody was used to detect the COOH-terminus of FPC. Strong positive immunoreactivity was seen only in the co-transfected sample, and weak immunoreactivity was detected in the FPC-C-Flag single-transfected sample, indicating that either exogenously transfected or endogenously expressed PC2 immunoprecipitates with FPC-C-Flag construct. This further confirms that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2. (H) Using the hAR-C2p antibody against the COOH-terminus of FPC to preincubate with FPC-C-Flag single-transfected protein lysates, positive immunoreactivity was seen only in the nonpreincubated co-IP sample, whereas the immunoreactivity was missing in the preincubated co-IP sample. Bar = 30 μm in B.
    Figure Legend Snippet: Molecular relationship between FPC and PC2. (A) Using the anti-PC2 mAb hPKD2-Cm1A11, Western blot of duplicate protein lysates from WT, Pkhd1 +/− , and Pkhd1 −/− E13.5 littermates showed a significant downregulation of PC2 in Pkhd1 −/− embryos, indicating that lack of FPC inhibits PC2 expression in vivo . An anti–β-actin antibody was used for a protein-loading control. (B) In comparison with the WT littermate (left), IHC staining with the anti-PC2 polyclonal antibody hPKD2-Cp showed a significant decrease in PC2 expression in the cortical region of the 1-mo-old Pkhd1 −/− kidney (right). (C) Duplicate lysates from E13.5 WT, Pkhd1 +/− , and Pkhd1 −/− littermates were used to perform a co-IP Western using the anti-FPC antibody hAR-Nm3G12 to IP and the anti-PC2 antibody hPKD2-Cm1A11 to detect PC2 expression. Positive immunoreactivity was seen in the WT embryo, and progressively reduced immunoreactivities were seen in the Pkhd1 +/− and Pkhd1 −/− littermates, suggesting that FPC binds to PC2 in vivo . (D) Lysates from E13.5 WT, Pkd2 +/− , and Pkd2 −/− littermates were used to perform a co-IP Western using the anti-PC2 antibody to IP and the anti-FPC antibody to detect FPC expression. Positive immunoreactivity was seen in the WT and Pkd2 +/− littermates, but no immunoreactivity was observed in the Pkd2 −/− littermate, providing further evidence that FPC physically interacts with PC2 in vivo . (E) There was no change in FPC expression in Western blot analysis among the WT and Pkd2 −/− littermates, indicating that the downregulation of PC2 does not affect FPC expression. (F) HA- and Flag-tagged expression vectors, in which the COOH-terminus of FPC (FPC-C-Flag) and the NH2-terminus of PC2 (PC2-N-HA) were constructed in-frame, were transiently co-transfected into HEK293 cells. Using an anti-Flag antibody to IP and an anti-HA antibody to detect the NH2-terminus of PC2, positive immunoreactivity was seen only in the co-transfected sample, indicating that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2 in vitro . (G) The same FPC-C-Flag expression vector was transiently co-transfected into HEK293 cells with an expression vector containing the human full-length PKD2 cDNA (PC2-Full). The anti-PC2 antibody hPKD2-Cm1A11 was used for IP and an anti-Flag antibody was used to detect the COOH-terminus of FPC. Strong positive immunoreactivity was seen only in the co-transfected sample, and weak immunoreactivity was detected in the FPC-C-Flag single-transfected sample, indicating that either exogenously transfected or endogenously expressed PC2 immunoprecipitates with FPC-C-Flag construct. This further confirms that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2. (H) Using the hAR-C2p antibody against the COOH-terminus of FPC to preincubate with FPC-C-Flag single-transfected protein lysates, positive immunoreactivity was seen only in the nonpreincubated co-IP sample, whereas the immunoreactivity was missing in the preincubated co-IP sample. Bar = 30 μm in B.

    Techniques Used: Western Blot, Expressing, In Vivo, Immunohistochemistry, Staining, Co-Immunoprecipitation Assay, Construct, Transfection, In Vitro, Plasmid Preparation

    30) Product Images from "Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function"

    Article Title: Fibrocystin/Polyductin Modulates Renal Tubular Formation by Regulating Polycystin-2 Expression and Function

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2007070770

    GFP expression in Pkhd1 mutant mice. (A) E15.5 Pkhd1 −/− kidney, liver, adrenal gland, and gastrointestinal tract stained with IHC using an anti-GFP polyclonal antibody. Positive signals were seen in the cortical adrenal cells (left arrow), periportal liver cells (right arrow), gastrointestinal tract (white box), and weakly positive staining was seen in the renal tubules (black box). (B) Higher magnification of the black box in A shows positive staining of the renal epithelia (arrow). (C) Higher magnification of the white box in A shows strong positive staining in the mucosal cells of the colon (arrow). (D) The differential interface contrast view showed GFP-positive staining (arrows) in the renal tubule epithelia of 4-mo-old Pkhd1 +/− (D, top) and Pkhd1 −/− (D, bottom) littermates. (E) Positive GFP staining was detected in an alveolar bronchiole of the lung in a 2-mo-old homozygous mouse (arrow). (F) Positive GFP was also seen in the tracheal epithelium of the same mouse (arrow). (G) GFP-positive staining (red) appeared in the ependymal cells lining the ventricles of the brain in a 2-mo-old homozygous mouse (arrow). Yo-pro (green) was used to stain the nuclei of cells. (H) GFP-positive staining (red) was observed in the diseased liver of a 2-mo-old homozygous mouse (arrows). (I) Cytokeratin 7–positive staining (green), a marker for epithelial cells, outlined the biliary epithelial structures (arrows). (J) The merged confocal image showed that GFP co-localized with cytokeratin 7. Bar = 30 μm in A; 15 μm in B and C; and 10 μm in D through J.
    Figure Legend Snippet: GFP expression in Pkhd1 mutant mice. (A) E15.5 Pkhd1 −/− kidney, liver, adrenal gland, and gastrointestinal tract stained with IHC using an anti-GFP polyclonal antibody. Positive signals were seen in the cortical adrenal cells (left arrow), periportal liver cells (right arrow), gastrointestinal tract (white box), and weakly positive staining was seen in the renal tubules (black box). (B) Higher magnification of the black box in A shows positive staining of the renal epithelia (arrow). (C) Higher magnification of the white box in A shows strong positive staining in the mucosal cells of the colon (arrow). (D) The differential interface contrast view showed GFP-positive staining (arrows) in the renal tubule epithelia of 4-mo-old Pkhd1 +/− (D, top) and Pkhd1 −/− (D, bottom) littermates. (E) Positive GFP staining was detected in an alveolar bronchiole of the lung in a 2-mo-old homozygous mouse (arrow). (F) Positive GFP was also seen in the tracheal epithelium of the same mouse (arrow). (G) GFP-positive staining (red) appeared in the ependymal cells lining the ventricles of the brain in a 2-mo-old homozygous mouse (arrow). Yo-pro (green) was used to stain the nuclei of cells. (H) GFP-positive staining (red) was observed in the diseased liver of a 2-mo-old homozygous mouse (arrows). (I) Cytokeratin 7–positive staining (green), a marker for epithelial cells, outlined the biliary epithelial structures (arrows). (J) The merged confocal image showed that GFP co-localized with cytokeratin 7. Bar = 30 μm in A; 15 μm in B and C; and 10 μm in D through J.

    Techniques Used: Expressing, Mutagenesis, Mouse Assay, Staining, Immunohistochemistry, Marker

    Molecular relationship between FPC and PC2. (A) Using the anti-PC2 mAb hPKD2-Cm1A11, Western blot of duplicate protein lysates from WT, Pkhd1 +/− , and Pkhd1 −/− E13.5 littermates showed a significant downregulation of PC2 in Pkhd1 −/− embryos, indicating that lack of FPC inhibits PC2 expression in vivo . An anti–β-actin antibody was used for a protein-loading control. (B) In comparison with the WT littermate (left), IHC staining with the anti-PC2 polyclonal antibody hPKD2-Cp showed a significant decrease in PC2 expression in the cortical region of the 1-mo-old Pkhd1 −/− kidney (right). (C) Duplicate lysates from E13.5 WT, Pkhd1 +/− , and Pkhd1 −/− littermates were used to perform a co-IP Western using the anti-FPC antibody hAR-Nm3G12 to IP and the anti-PC2 antibody hPKD2-Cm1A11 to detect PC2 expression. Positive immunoreactivity was seen in the WT embryo, and progressively reduced immunoreactivities were seen in the Pkhd1 +/− and Pkhd1 −/− littermates, suggesting that FPC binds to PC2 in vivo . (D) Lysates from E13.5 WT, Pkd2 +/− , and Pkd2 −/− littermates were used to perform a co-IP Western using the anti-PC2 antibody to IP and the anti-FPC antibody to detect FPC expression. Positive immunoreactivity was seen in the WT and Pkd2 +/− littermates, but no immunoreactivity was observed in the Pkd2 −/− littermate, providing further evidence that FPC physically interacts with PC2 in vivo . (E) There was no change in FPC expression in Western blot analysis among the WT and Pkd2 −/− littermates, indicating that the downregulation of PC2 does not affect FPC expression. (F) HA- and Flag-tagged expression vectors, in which the COOH-terminus of FPC (FPC-C-Flag) and the NH2-terminus of PC2 (PC2-N-HA) were constructed in-frame, were transiently co-transfected into HEK293 cells. Using an anti-Flag antibody to IP and an anti-HA antibody to detect the NH2-terminus of PC2, positive immunoreactivity was seen only in the co-transfected sample, indicating that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2 in vitro . (G) The same FPC-C-Flag expression vector was transiently co-transfected into HEK293 cells with an expression vector containing the human full-length PKD2 cDNA (PC2-Full). The anti-PC2 antibody hPKD2-Cm1A11 was used for IP and an anti-Flag antibody was used to detect the COOH-terminus of FPC. Strong positive immunoreactivity was seen only in the co-transfected sample, and weak immunoreactivity was detected in the FPC-C-Flag single-transfected sample, indicating that either exogenously transfected or endogenously expressed PC2 immunoprecipitates with FPC-C-Flag construct. This further confirms that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2. (H) Using the hAR-C2p antibody against the COOH-terminus of FPC to preincubate with FPC-C-Flag single-transfected protein lysates, positive immunoreactivity was seen only in the nonpreincubated co-IP sample, whereas the immunoreactivity was missing in the preincubated co-IP sample. Bar = 30 μm in B.
    Figure Legend Snippet: Molecular relationship between FPC and PC2. (A) Using the anti-PC2 mAb hPKD2-Cm1A11, Western blot of duplicate protein lysates from WT, Pkhd1 +/− , and Pkhd1 −/− E13.5 littermates showed a significant downregulation of PC2 in Pkhd1 −/− embryos, indicating that lack of FPC inhibits PC2 expression in vivo . An anti–β-actin antibody was used for a protein-loading control. (B) In comparison with the WT littermate (left), IHC staining with the anti-PC2 polyclonal antibody hPKD2-Cp showed a significant decrease in PC2 expression in the cortical region of the 1-mo-old Pkhd1 −/− kidney (right). (C) Duplicate lysates from E13.5 WT, Pkhd1 +/− , and Pkhd1 −/− littermates were used to perform a co-IP Western using the anti-FPC antibody hAR-Nm3G12 to IP and the anti-PC2 antibody hPKD2-Cm1A11 to detect PC2 expression. Positive immunoreactivity was seen in the WT embryo, and progressively reduced immunoreactivities were seen in the Pkhd1 +/− and Pkhd1 −/− littermates, suggesting that FPC binds to PC2 in vivo . (D) Lysates from E13.5 WT, Pkd2 +/− , and Pkd2 −/− littermates were used to perform a co-IP Western using the anti-PC2 antibody to IP and the anti-FPC antibody to detect FPC expression. Positive immunoreactivity was seen in the WT and Pkd2 +/− littermates, but no immunoreactivity was observed in the Pkd2 −/− littermate, providing further evidence that FPC physically interacts with PC2 in vivo . (E) There was no change in FPC expression in Western blot analysis among the WT and Pkd2 −/− littermates, indicating that the downregulation of PC2 does not affect FPC expression. (F) HA- and Flag-tagged expression vectors, in which the COOH-terminus of FPC (FPC-C-Flag) and the NH2-terminus of PC2 (PC2-N-HA) were constructed in-frame, were transiently co-transfected into HEK293 cells. Using an anti-Flag antibody to IP and an anti-HA antibody to detect the NH2-terminus of PC2, positive immunoreactivity was seen only in the co-transfected sample, indicating that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2 in vitro . (G) The same FPC-C-Flag expression vector was transiently co-transfected into HEK293 cells with an expression vector containing the human full-length PKD2 cDNA (PC2-Full). The anti-PC2 antibody hPKD2-Cm1A11 was used for IP and an anti-Flag antibody was used to detect the COOH-terminus of FPC. Strong positive immunoreactivity was seen only in the co-transfected sample, and weak immunoreactivity was detected in the FPC-C-Flag single-transfected sample, indicating that either exogenously transfected or endogenously expressed PC2 immunoprecipitates with FPC-C-Flag construct. This further confirms that the COOH-terminus of FPC physically interacts with the NH2-terminus of PC2. (H) Using the hAR-C2p antibody against the COOH-terminus of FPC to preincubate with FPC-C-Flag single-transfected protein lysates, positive immunoreactivity was seen only in the nonpreincubated co-IP sample, whereas the immunoreactivity was missing in the preincubated co-IP sample. Bar = 30 μm in B.

    Techniques Used: Western Blot, Expressing, In Vivo, Immunohistochemistry, Staining, Co-Immunoprecipitation Assay, Construct, Transfection, In Vitro, Plasmid Preparation

    31) Product Images from "Nestin Promotes the Phosphorylation-dependent Disassembly of Vimentin Intermediate Filaments During Mitosis"

    Article Title: Nestin Promotes the Phosphorylation-dependent Disassembly of Vimentin Intermediate Filaments During Mitosis

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E02-08-0545

    Nestin expression in cultured cell types. (A) Immunoblotting analysis of IF-enriched cytoskeletal samples from seven cultured cell lines with an affinity-purified polyclonal nestin antibody (see Materials and Methods). In cell types such as C6–2, vimentin (VIM, red) and nestin (NES, green) are present in a nonfilamentous punctate pattern during mitosis (B and C). The mitotic state (metaphase) of this cell is indicated by the presence of condensed chromosomes (blue). In cell types such as 3T3 (D and E) and CV-1 (F and G) that do not express nestin, the vimentin IFs remain filamentous, as shown in an anaphase 3T3 cell (D and E) and a metaphase/early anaphase CV-1 cell (F and G). Bar for B–E, F, and G, 5 μm.
    Figure Legend Snippet: Nestin expression in cultured cell types. (A) Immunoblotting analysis of IF-enriched cytoskeletal samples from seven cultured cell lines with an affinity-purified polyclonal nestin antibody (see Materials and Methods). In cell types such as C6–2, vimentin (VIM, red) and nestin (NES, green) are present in a nonfilamentous punctate pattern during mitosis (B and C). The mitotic state (metaphase) of this cell is indicated by the presence of condensed chromosomes (blue). In cell types such as 3T3 (D and E) and CV-1 (F and G) that do not express nestin, the vimentin IFs remain filamentous, as shown in an anaphase 3T3 cell (D and E) and a metaphase/early anaphase CV-1 cell (F and G). Bar for B–E, F, and G, 5 μm.

    Techniques Used: Expressing, Cell Culture, Affinity Purification

    32) Product Images from "Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins"

    Article Title: Recruitment of cortexillin into the cleavage furrow is controlled by Rac1 and IQGAP-related proteins

    Journal: The EMBO Journal

    doi: 10.1093/emboj/20.14.3705

    Fig. 3. DGAP1 or GAPA link CI into a quarternary complex with activated Rac1A. ( A ) DGAP1 and CI form a complex preferentially with activated Rac1A in vitro . Western blot of lysates prepared from AX2 cells or from mutants lacking either DGAP1 or CI labeled with anti-DGAP1 mAb 216-394-1 and anti-CI mAb 241-438-1 (left). The glutathione–Sepharose bound GST-control or GST–Rac1A fusion proteins were loaded with either GDP or GTPγS and were incubated with the lysates indicated. Bound proteins were eluted with SDS sample buffer, and aliquots analyzed by western blotting using the same antibodies. ( B ). ( C ) Western blot of immunoprecipitates obtained from AX2 and DGAP1-null-derived transformants that express wild-type (Rac1A-WT), constitutively activated (Rac1A-V12) or constitutively inactivated (Rac1A-N17) Rac1A, N-terminally fused to GFP. The GFP fusion proteins were immunoprecipitated with polyclonal anti-GFP antibodies, and the precipitates analyzed by triple labeling with anti-GFP, anti-DGAP1 and anti-CI monoclonal antibodies (top). A parallel blot was labeled for CII (bottom). DGAP1 and both cortexillins were exclusively co-immunoprecipitated with Rac1A-WT and Rac1A-V12, demonstrating that the complex is only formed with the activated form of the GTPase in vivo . A similar complex containing activated Rac1A, both cortexillins and another protein was formed in DGAP1-null-derived transformants. ( D ) To visualize the protein required for complex formation in DGAP1-null mutants, a parallel gel was developed with silver. White arrowheads indicate the position of DGAP1, and black arrowheads denote the position of a 100 kDa protein isolated from DGAP1-null cells, which displayed similar binding properties as DGAP1. This protein was identified by peptide fingerprinting as GAPA.
    Figure Legend Snippet: Fig. 3. DGAP1 or GAPA link CI into a quarternary complex with activated Rac1A. ( A ) DGAP1 and CI form a complex preferentially with activated Rac1A in vitro . Western blot of lysates prepared from AX2 cells or from mutants lacking either DGAP1 or CI labeled with anti-DGAP1 mAb 216-394-1 and anti-CI mAb 241-438-1 (left). The glutathione–Sepharose bound GST-control or GST–Rac1A fusion proteins were loaded with either GDP or GTPγS and were incubated with the lysates indicated. Bound proteins were eluted with SDS sample buffer, and aliquots analyzed by western blotting using the same antibodies. ( B ). ( C ) Western blot of immunoprecipitates obtained from AX2 and DGAP1-null-derived transformants that express wild-type (Rac1A-WT), constitutively activated (Rac1A-V12) or constitutively inactivated (Rac1A-N17) Rac1A, N-terminally fused to GFP. The GFP fusion proteins were immunoprecipitated with polyclonal anti-GFP antibodies, and the precipitates analyzed by triple labeling with anti-GFP, anti-DGAP1 and anti-CI monoclonal antibodies (top). A parallel blot was labeled for CII (bottom). DGAP1 and both cortexillins were exclusively co-immunoprecipitated with Rac1A-WT and Rac1A-V12, demonstrating that the complex is only formed with the activated form of the GTPase in vivo . A similar complex containing activated Rac1A, both cortexillins and another protein was formed in DGAP1-null-derived transformants. ( D ) To visualize the protein required for complex formation in DGAP1-null mutants, a parallel gel was developed with silver. White arrowheads indicate the position of DGAP1, and black arrowheads denote the position of a 100 kDa protein isolated from DGAP1-null cells, which displayed similar binding properties as DGAP1. This protein was identified by peptide fingerprinting as GAPA.

    Techniques Used: In Vitro, Western Blot, Labeling, Incubation, Derivative Assay, Immunoprecipitation, In Vivo, Isolation, Binding Assay

    Fig. 1. DGAP1 interacts with the C-terminus of CI. (A–C) DGAP1 specifically co-immunoprecipitates with CI. ( A ) Western blot of total cellular proteins from the cell lines used for the immunoprecipitation labeled with anti-GFP mAb 264-449-2. ( B ) After immunoprecipitation with anti-GFP polyclonal antibodies, bound proteins were resolved by SDS–PAGE and stained with silver. The 95 kDa protein specifically co-immunoprecipitated with GFP–CI was identified as DGAP1. ( C ) Western blotting of the immunoprecipitates with anti-DGAP1 mAb 216-394-1. (D–F) DGAP1 interacts with the CI isoform. ( D ) Western blot of total cellular proteins from the cell lines used for the immunoprecipitation labeled with anti-GFP mAb 264-449-2. ( E ) Silver-stained gel of the immunoprecipitates obtained with anti-GFP polyclonal antibodies. ( F ) Immunoblot of the immunoprecipitates labeled with anti-DGAP1 mAb 216-394-1. DGAP1 was only co-immunoprecipitated from lysates of cell lines expressing either endogenous or GFP-tagged cortexillin I. (G–I) Mapping of the DGAP1-binding region on CI. ( G ) Western blot of AX2 and AX2-GFP control cells and of CI – cells expressing full-length (1–444) CI, the N-terminal actin-binding site (1–233), the complete C-terminal domain (352–444), and the C-terminal domain without the PIP 2 -binding site (352–435) fused to GFP. The blot was labeled with anti-GFP mAb 264-449-2. ( H ) Silver-stained gel of the proteins precipitated with anti-GFP polyclonal antibodies. ( J ) Western blot of the immunoprecipitates labeled with anti-DGAP1 mAb 216-394-1.
    Figure Legend Snippet: Fig. 1. DGAP1 interacts with the C-terminus of CI. (A–C) DGAP1 specifically co-immunoprecipitates with CI. ( A ) Western blot of total cellular proteins from the cell lines used for the immunoprecipitation labeled with anti-GFP mAb 264-449-2. ( B ) After immunoprecipitation with anti-GFP polyclonal antibodies, bound proteins were resolved by SDS–PAGE and stained with silver. The 95 kDa protein specifically co-immunoprecipitated with GFP–CI was identified as DGAP1. ( C ) Western blotting of the immunoprecipitates with anti-DGAP1 mAb 216-394-1. (D–F) DGAP1 interacts with the CI isoform. ( D ) Western blot of total cellular proteins from the cell lines used for the immunoprecipitation labeled with anti-GFP mAb 264-449-2. ( E ) Silver-stained gel of the immunoprecipitates obtained with anti-GFP polyclonal antibodies. ( F ) Immunoblot of the immunoprecipitates labeled with anti-DGAP1 mAb 216-394-1. DGAP1 was only co-immunoprecipitated from lysates of cell lines expressing either endogenous or GFP-tagged cortexillin I. (G–I) Mapping of the DGAP1-binding region on CI. ( G ) Western blot of AX2 and AX2-GFP control cells and of CI – cells expressing full-length (1–444) CI, the N-terminal actin-binding site (1–233), the complete C-terminal domain (352–444), and the C-terminal domain without the PIP 2 -binding site (352–435) fused to GFP. The blot was labeled with anti-GFP mAb 264-449-2. ( H ) Silver-stained gel of the proteins precipitated with anti-GFP polyclonal antibodies. ( J ) Western blot of the immunoprecipitates labeled with anti-DGAP1 mAb 216-394-1.

    Techniques Used: Western Blot, Immunoprecipitation, Labeling, SDS Page, Staining, Expressing, Binding Assay

    33) Product Images from "Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite"

    Article Title: Intramembrane proteolysis mediates shedding of a key adhesin during erythrocyte invasion by the malaria parasite

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200604136

    PfROM4 is a merozoite plasma membrane protein. (A) Western blot showing extracts of parental 3D7 and 3D7HAROM4 synth probed with mAb 3F10 (anti-HA) and polyclonal anti-AMA1 (loading control). The size of the anti-HA–reactive species detected only in 3D7HAROM4 synth is close to the predicted mass (90.4 kD) of HA-tagged PfROM4. (B) IFA of mature nonsegmented (top) and fully segmented (bottom) schizonts of 3D7HAROM4 synth , dual labeled with mAb 3F10 (anti-HA; green) and mAb 1E1 (anti-MSP1; red). The DAPI signal (blue) is not included in merged images for clarity. Bar, 5 μm.
    Figure Legend Snippet: PfROM4 is a merozoite plasma membrane protein. (A) Western blot showing extracts of parental 3D7 and 3D7HAROM4 synth probed with mAb 3F10 (anti-HA) and polyclonal anti-AMA1 (loading control). The size of the anti-HA–reactive species detected only in 3D7HAROM4 synth is close to the predicted mass (90.4 kD) of HA-tagged PfROM4. (B) IFA of mature nonsegmented (top) and fully segmented (bottom) schizonts of 3D7HAROM4 synth , dual labeled with mAb 3F10 (anti-HA; green) and mAb 1E1 (anti-MSP1; red). The DAPI signal (blue) is not included in merged images for clarity. Bar, 5 μm.

    Techniques Used: Western Blot, Immunofluorescence, Labeling

    34) Product Images from "Diaphanous gene mutation affects spiral cleavage and chirality in snails"

    Article Title: Diaphanous gene mutation affects spiral cleavage and chirality in snails

    Journal: Scientific Reports

    doi: 10.1038/srep34809

    Generation of an LsDia1-specific antibody and developmental expression of LsDia1 protein. ( a ) Comparison of the amino acid sequences of the C-terminal region between LsDia1 (residues 1054–1068) and LsDia2 (residues 1066–1080), which were selected as a target site for making anti-LsDia1 antibody. Underlined sequence indicates the recognition site of anti-LsDia1 polyclonal antibodies. ( b ) Western blotting analyses showed that LsDia1 protein is present in the dextral embryos from 1-cell immediately after oviposition to the blastula stage, but is not detectable for the sinistral embryos even at the 1-cell stage prior to 1st pb extrusion. LsDia1 and β-Tubulin levels were analyzed sequentially using the same blot. The whole images of blot showing the single band of correct MW for LsDia1 and β-Tubulin, respectively, are presented in Supplementary Figure S4 . ( c ) Presence and absence of LsDia1 protein in early stage embryos of the congenic (F10) dd strain (1–4-cell) and DD strain (1-cell), respectively.
    Figure Legend Snippet: Generation of an LsDia1-specific antibody and developmental expression of LsDia1 protein. ( a ) Comparison of the amino acid sequences of the C-terminal region between LsDia1 (residues 1054–1068) and LsDia2 (residues 1066–1080), which were selected as a target site for making anti-LsDia1 antibody. Underlined sequence indicates the recognition site of anti-LsDia1 polyclonal antibodies. ( b ) Western blotting analyses showed that LsDia1 protein is present in the dextral embryos from 1-cell immediately after oviposition to the blastula stage, but is not detectable for the sinistral embryos even at the 1-cell stage prior to 1st pb extrusion. LsDia1 and β-Tubulin levels were analyzed sequentially using the same blot. The whole images of blot showing the single band of correct MW for LsDia1 and β-Tubulin, respectively, are presented in Supplementary Figure S4 . ( c ) Presence and absence of LsDia1 protein in early stage embryos of the congenic (F10) dd strain (1–4-cell) and DD strain (1-cell), respectively.

    Techniques Used: Expressing, Sequencing, Western Blot

    35) Product Images from "Comparative Analysis of B- and T-Cell Epitopes of Mycobacterium leprae and Mycobacterium tuberculosis Culture Filtrate Protein 10 "

    Article Title: Comparative Analysis of B- and T-Cell Epitopes of Mycobacterium leprae and Mycobacterium tuberculosis Culture Filtrate Protein 10

    Journal: Infection and Immunity

    doi: 10.1128/IAI.72.6.3161-3170.2004

    Immunohistological staining of M. leprae -infected nu/nu mouse footpad sections with polyclonal rabbit anti- M. leprae CFP-10. Formalin-fixed tissue embedded in paraffin was cut into 5-μm sections and stained by conventional methods. (A) Kinyoun method for acid-fast staining, showing specific acid-fast staining of bacilli within (arrow) and outside (arrowhead) necrotic cell pockets. Magnification, ×200. (B) Hematoxylin and eosin staining, showing a pocket of necrotic cells (arrow) and extensive infiltration of foamy-like macrophages (arrowhead). Magnification, ×200. (C and D) Immunohistochemistry with a rabbit polyclonal antibody against M. leprae CFP-10, indicated by dark red staining in the center of cells within the granuloma (arrows). Magnification, ×400.
    Figure Legend Snippet: Immunohistological staining of M. leprae -infected nu/nu mouse footpad sections with polyclonal rabbit anti- M. leprae CFP-10. Formalin-fixed tissue embedded in paraffin was cut into 5-μm sections and stained by conventional methods. (A) Kinyoun method for acid-fast staining, showing specific acid-fast staining of bacilli within (arrow) and outside (arrowhead) necrotic cell pockets. Magnification, ×200. (B) Hematoxylin and eosin staining, showing a pocket of necrotic cells (arrow) and extensive infiltration of foamy-like macrophages (arrowhead). Magnification, ×200. (C and D) Immunohistochemistry with a rabbit polyclonal antibody against M. leprae CFP-10, indicated by dark red staining in the center of cells within the granuloma (arrows). Magnification, ×400.

    Techniques Used: Staining, Infection, Immunohistochemistry

    Reactivities of polyclonal anti- M. leprae CFP-10 and anti- M. tuberculosis CFP-10 with homologous and heterologous CFP-10 as determined by ELISA. M . l . anti- M . l . CFP-10, M. leprae anti- M. leprae CFP-10; M . l . anti- Mtb CFP-10, M. leprae anti- M. tuberculosis CFP-10; Mtb anti- Mtb CFP-10, M. tuberculosis anti- M. tuberculosis CFP-10; Mtb anti- M.l. CFP-10, M. tuberculosis anti- M. leprae CFP-10; Ab, antibody; O.D. 405 nm, optical density at 405 nm.
    Figure Legend Snippet: Reactivities of polyclonal anti- M. leprae CFP-10 and anti- M. tuberculosis CFP-10 with homologous and heterologous CFP-10 as determined by ELISA. M . l . anti- M . l . CFP-10, M. leprae anti- M. leprae CFP-10; M . l . anti- Mtb CFP-10, M. leprae anti- M. tuberculosis CFP-10; Mtb anti- Mtb CFP-10, M. tuberculosis anti- M. tuberculosis CFP-10; Mtb anti- M.l. CFP-10, M. tuberculosis anti- M. leprae CFP-10; Ab, antibody; O.D. 405 nm, optical density at 405 nm.

    Techniques Used: Enzyme-linked Immunosorbent Assay

    Western blot of M. leprae purified rCFP-10 (lanes 1, 3, 5, 7, and 9) and M. tuberculosis purified rCFP-10 (lanes 2, 4, 6, 8, and 10) with MAbs and polyclonal antibodies. Lanes 1 and 2 contained equivalent amounts of the proteins electrophoresed on a 15% polyacrylamide gel and silver stained; lanes 3 and 4 were probed with rabbit polyclonal antiserum against M. leprae CFP-10 (rabbit α Ml CFP-10) (1:10,000 dilution); lanes 5 and 6 were probed with mouse polyclonal antiserum against M. leprae CFP-10 (mouse α Ml CFP-10) (1:2,500 dilution); lanes 7 and 8 were probed with mouse MAb 3A10.2C8 specific for peptides p5 and p6 (1:20 dilution of culture supernatant); and lanes 9 and 10 were probed with the mouse polyclonal antiserum against M. tuberculosis CFP-10 (mouse α Mtb CFP-10) (1:2,500 dilution).
    Figure Legend Snippet: Western blot of M. leprae purified rCFP-10 (lanes 1, 3, 5, 7, and 9) and M. tuberculosis purified rCFP-10 (lanes 2, 4, 6, 8, and 10) with MAbs and polyclonal antibodies. Lanes 1 and 2 contained equivalent amounts of the proteins electrophoresed on a 15% polyacrylamide gel and silver stained; lanes 3 and 4 were probed with rabbit polyclonal antiserum against M. leprae CFP-10 (rabbit α Ml CFP-10) (1:10,000 dilution); lanes 5 and 6 were probed with mouse polyclonal antiserum against M. leprae CFP-10 (mouse α Ml CFP-10) (1:2,500 dilution); lanes 7 and 8 were probed with mouse MAb 3A10.2C8 specific for peptides p5 and p6 (1:20 dilution of culture supernatant); and lanes 9 and 10 were probed with the mouse polyclonal antiserum against M. tuberculosis CFP-10 (mouse α Mtb CFP-10) (1:2,500 dilution).

    Techniques Used: Western Blot, Purification, Staining

    36) Product Images from "Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation"

    Article Title: Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00798-13

    The influence of TopA levels on DNA supercoiling in vivo . (A) The level of the topA transcripts in noninduced PS01 compared with that in the wild-type (M145), as quantified by qRT-PCR. (B) The level of TopA proteins in the PS01 strain subjected to thiostrepton induction, as quantified by Western blotting using anti-TopA polyclonal antibodies. (C) Agarose gel electrophoresis showing the topoisomer distribution of pWHM3Hyg plasmids isolated from PS01 (liquid culture; 48 h in 79 medium) induced with different concentrations of thiostrepton (0 to 10 μg/ml). WT, wild type; RQ, relative quantification; conc, concentration.
    Figure Legend Snippet: The influence of TopA levels on DNA supercoiling in vivo . (A) The level of the topA transcripts in noninduced PS01 compared with that in the wild-type (M145), as quantified by qRT-PCR. (B) The level of TopA proteins in the PS01 strain subjected to thiostrepton induction, as quantified by Western blotting using anti-TopA polyclonal antibodies. (C) Agarose gel electrophoresis showing the topoisomer distribution of pWHM3Hyg plasmids isolated from PS01 (liquid culture; 48 h in 79 medium) induced with different concentrations of thiostrepton (0 to 10 μg/ml). WT, wild type; RQ, relative quantification; conc, concentration.

    Techniques Used: In Vivo, Quantitative RT-PCR, Western Blot, Agarose Gel Electrophoresis, Isolation, Concentration Assay

    37) Product Images from "Cell Membrane Is Impaired, Accompanied by Enhanced Type III Secretion System Expression in Yersinia pestis Deficient in RovA Regulator"

    Article Title: Cell Membrane Is Impaired, Accompanied by Enhanced Type III Secretion System Expression in Yersinia pestis Deficient in RovA Regulator

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0012840

    Ability of RovA to bind to promoters of lcrF and lcrG of T3SS. The upstream regions of lcrG, yscA, lcrF and psaE were amplified by PCR and used as target DNA probes in EMSA. The [γ-32P]-labeled target DNA probes were incubated with or without increasing amounts of purified His-RovA protein (lanes 1–4). Three controls were included in each EMSA experiment as indicated: 1) non-specific probe competitor (unlabeled DNA probe containing promoter of a gene that was shown to be not affected by rovA mutation); 2) specific probe competitor (unlabeled DNA probe containing promoter region of the investigated gene); and 3) unrelated proteins (rabbit anti-F1-protein polyclonal antibody).
    Figure Legend Snippet: Ability of RovA to bind to promoters of lcrF and lcrG of T3SS. The upstream regions of lcrG, yscA, lcrF and psaE were amplified by PCR and used as target DNA probes in EMSA. The [γ-32P]-labeled target DNA probes were incubated with or without increasing amounts of purified His-RovA protein (lanes 1–4). Three controls were included in each EMSA experiment as indicated: 1) non-specific probe competitor (unlabeled DNA probe containing promoter of a gene that was shown to be not affected by rovA mutation); 2) specific probe competitor (unlabeled DNA probe containing promoter region of the investigated gene); and 3) unrelated proteins (rabbit anti-F1-protein polyclonal antibody).

    Techniques Used: Amplification, Polymerase Chain Reaction, Labeling, Incubation, Purification, Mutagenesis

    Influence of RovA on Yop expression and secretion. Bacterial strains were grown in TMH medium without calcium at 26°C to an OD 600 of ∼1.0 and then transferred to 37°C for 3 h to induce the expression and secretion of Yop proteins. TCA was used to precipitate proteins from the culture supernatants. The bacterial cell pellets were separated by SDS-PAGE, and specific proteins were detected using rabbit polyclonal antibodies against YopE, YopJ and YopM (A). C and S stand for proteins separated from the cell pellet and bacterial culture supernatant, respectively. In lane 4, the band right below the YopM band could be the degraded product of YopM, and a large amount of this product could be stably detected in the ΔrovA mutant; however, it was much less in the wild type and the ΔrovA mutant complemented with ΔrovA- pAraRovA. Densitometry analysis of Western blots was performed using TotalLab software, and the numbers indicate the ratios of the densitometry values from each lane to lane 1 of each row (B). W: wild type strain; M: mutant strain ΔrovA ; C: complementary ΔrovA- pAraRovA strain.
    Figure Legend Snippet: Influence of RovA on Yop expression and secretion. Bacterial strains were grown in TMH medium without calcium at 26°C to an OD 600 of ∼1.0 and then transferred to 37°C for 3 h to induce the expression and secretion of Yop proteins. TCA was used to precipitate proteins from the culture supernatants. The bacterial cell pellets were separated by SDS-PAGE, and specific proteins were detected using rabbit polyclonal antibodies against YopE, YopJ and YopM (A). C and S stand for proteins separated from the cell pellet and bacterial culture supernatant, respectively. In lane 4, the band right below the YopM band could be the degraded product of YopM, and a large amount of this product could be stably detected in the ΔrovA mutant; however, it was much less in the wild type and the ΔrovA mutant complemented with ΔrovA- pAraRovA. Densitometry analysis of Western blots was performed using TotalLab software, and the numbers indicate the ratios of the densitometry values from each lane to lane 1 of each row (B). W: wild type strain; M: mutant strain ΔrovA ; C: complementary ΔrovA- pAraRovA strain.

    Techniques Used: Expressing, SDS Page, Stable Transfection, Mutagenesis, Western Blot, Software

    RovA expression in Y. pestis strain 201, the ΔrovA mutant and ΔrovA- pAraRovA. Overnight cultures of bacterial strains grown in BHI at 26°C were harvested and whole cell lysates were separated by SDS-PAGE. The expression of rovA was detected by Western blotting using a rabbit polyclonal antibody against His-tagged RovA. For the induction of RovA expression in ΔrovA- pAraRovA, arabinose was added to the culture medium at the indicated concentration.
    Figure Legend Snippet: RovA expression in Y. pestis strain 201, the ΔrovA mutant and ΔrovA- pAraRovA. Overnight cultures of bacterial strains grown in BHI at 26°C were harvested and whole cell lysates were separated by SDS-PAGE. The expression of rovA was detected by Western blotting using a rabbit polyclonal antibody against His-tagged RovA. For the induction of RovA expression in ΔrovA- pAraRovA, arabinose was added to the culture medium at the indicated concentration.

    Techniques Used: Expressing, Mutagenesis, SDS Page, Western Blot, Concentration Assay

    38) Product Images from "Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma"

    Article Title: Loss of connective tissue growth factor as an unfavorable prognosis factor activates miR-18b by PI3K/AKT/C-Jun and C-Myc and promotes cell growth in nasopharyngeal carcinoma

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2013.153

    Stable suppression of CTGF expression stimulated the proliferation of NPC cells and speeded up the transition of cell cycle from G1 to S. ( a ) (A1) Polyclonal cells of lentivirus-mediated shRNA-CTGF-A and B, and PLV-Ctr were screened by GFP using FACS cytometry assay. (A2) Expression of CTGF was suppressed in shRNA-CTGF-A and B compared with PLV-Ctr cells by western blot. ( b ) In vitro proliferative ability of NPC cells was significantly restored in CTGF-suppressed cells compared with PLV-Ctr cells by MTT assay. ( c ) When compared with PLV-Ctr cells, tumor weight of shRNA-CTGF-A and B cells was markedly increased in vivo . ( d ) When compared with PLV-Ctr cells, tumor volume of shRNA-CTGF-A and B cells was markedly increased in vivo . ( e ) Downregulated CTGF expression stimulated cell cycle progression from G1 to S in shRNA-CTGF-A and B cells ( P
    Figure Legend Snippet: Stable suppression of CTGF expression stimulated the proliferation of NPC cells and speeded up the transition of cell cycle from G1 to S. ( a ) (A1) Polyclonal cells of lentivirus-mediated shRNA-CTGF-A and B, and PLV-Ctr were screened by GFP using FACS cytometry assay. (A2) Expression of CTGF was suppressed in shRNA-CTGF-A and B compared with PLV-Ctr cells by western blot. ( b ) In vitro proliferative ability of NPC cells was significantly restored in CTGF-suppressed cells compared with PLV-Ctr cells by MTT assay. ( c ) When compared with PLV-Ctr cells, tumor weight of shRNA-CTGF-A and B cells was markedly increased in vivo . ( d ) When compared with PLV-Ctr cells, tumor volume of shRNA-CTGF-A and B cells was markedly increased in vivo . ( e ) Downregulated CTGF expression stimulated cell cycle progression from G1 to S in shRNA-CTGF-A and B cells ( P

    Techniques Used: Expressing, shRNA, FACS, Cytometry, Western Blot, In Vitro, MTT Assay, In Vivo

    39) Product Images from "Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes"

    Article Title: Arginase Is Essential for Survival of Leishmania donovani Promastigotes but Not Intracellular Amastigotes

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00554-16

    Localization of ARG and argΔAKL. Wild-type (A to C), Δ arg (D to F), Δ arg [ ARG ] (G to I), or Δ arg [ arg ΔAKL] (J to L) L. donovani promastigotes were subjected to immunofluorescence analysis using rabbit anti-ARG polyclonal antibodies (A, D, G, and J). Goat anti-rabbit Oregon green-conjugated secondary antibody was used to detect the ARG primary antibodies. Parasites were also stained with DAPI (B, E, H, and K) and photographed using differential interference contrast (DIC) (C, F, I, and L).
    Figure Legend Snippet: Localization of ARG and argΔAKL. Wild-type (A to C), Δ arg (D to F), Δ arg [ ARG ] (G to I), or Δ arg [ arg ΔAKL] (J to L) L. donovani promastigotes were subjected to immunofluorescence analysis using rabbit anti-ARG polyclonal antibodies (A, D, G, and J). Goat anti-rabbit Oregon green-conjugated secondary antibody was used to detect the ARG primary antibodies. Parasites were also stained with DAPI (B, E, H, and K) and photographed using differential interference contrast (DIC) (C, F, I, and L).

    Techniques Used: Immunofluorescence, Staining

    Genotypic and phenotypic analysis of genetically manipulated parasites. (A, B, and C) Genomic DNA from the following parasite strains was used as the template: lanes 1, wild type; lanes 2, ARG/arg heterozygote; lanes 3, Δ arg mutant 1; lanes 4, Δ arg mutant 2; lanes 5, Δ arg [ ARG ] mutant; lanes 6, Δ arg [ arg ΔAKL] mutant. (A) Primers designed to encompass the coding region were used to amplify the ARG gene. (B) The forward primer sequence was located in a region upstream of the 5′ flanking sequence (upstream of the targeting construct), and the reverse primer sequence was located within the hygromycin resistance gene. (C) The same sense primer as that used for panel B was used, and the antisense primer sequence was located within the phleomycin resistance gene. (D) Western blot analyses were performed with cell extracts prepared from wild-type, ARG/arg heterozygote, Δ arg mutant 1, Δ arg mutant 2, Δ arg [ ARG ], and Δ arg [ arg ΔAKL] parasites. Parasites were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. mexicana ARG and a commercial monoclonal antibody that recognizes tubulin. The tubulin antibody was employed to verify equal loading of protein on all lanes.
    Figure Legend Snippet: Genotypic and phenotypic analysis of genetically manipulated parasites. (A, B, and C) Genomic DNA from the following parasite strains was used as the template: lanes 1, wild type; lanes 2, ARG/arg heterozygote; lanes 3, Δ arg mutant 1; lanes 4, Δ arg mutant 2; lanes 5, Δ arg [ ARG ] mutant; lanes 6, Δ arg [ arg ΔAKL] mutant. (A) Primers designed to encompass the coding region were used to amplify the ARG gene. (B) The forward primer sequence was located in a region upstream of the 5′ flanking sequence (upstream of the targeting construct), and the reverse primer sequence was located within the hygromycin resistance gene. (C) The same sense primer as that used for panel B was used, and the antisense primer sequence was located within the phleomycin resistance gene. (D) Western blot analyses were performed with cell extracts prepared from wild-type, ARG/arg heterozygote, Δ arg mutant 1, Δ arg mutant 2, Δ arg [ ARG ], and Δ arg [ arg ΔAKL] parasites. Parasites were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. mexicana ARG and a commercial monoclonal antibody that recognizes tubulin. The tubulin antibody was employed to verify equal loading of protein on all lanes.

    Techniques Used: Mutagenesis, Sequencing, Construct, Western Blot, SDS Page

    Ornithine requirement for Δ arg [ ODC ] promastigotes. (A) Western blot analysis was performed with cell lysates prepared from wild-type parasites, the parental Δ arg line, and the Δ arg [ ODC ] ODC overproducer strain. Parasite lysates were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. donovani ODC and an anti-tubulin antibody as a loading control. (B) Growth phenotypes of Δ arg (gray squares) and Δ arg [ ODC ] (black triangles) promastigotes were established in increasing concentrations of ornithine. Parasites were incubated at 5 × 10 5 parasites/ml, and percent proliferation was evaluated after 5 days via the ability of parasites to convert resazurin to resorufin as assessed by fluorescence, and readings obtained with the highest supplement concentrations were equated with 100% proliferation. The experiments were set up in duplicate and repeated three times with similar results.
    Figure Legend Snippet: Ornithine requirement for Δ arg [ ODC ] promastigotes. (A) Western blot analysis was performed with cell lysates prepared from wild-type parasites, the parental Δ arg line, and the Δ arg [ ODC ] ODC overproducer strain. Parasite lysates were fractioned by SDS-PAGE and the blot probed with polyclonal antibodies against L. donovani ODC and an anti-tubulin antibody as a loading control. (B) Growth phenotypes of Δ arg (gray squares) and Δ arg [ ODC ] (black triangles) promastigotes were established in increasing concentrations of ornithine. Parasites were incubated at 5 × 10 5 parasites/ml, and percent proliferation was evaluated after 5 days via the ability of parasites to convert resazurin to resorufin as assessed by fluorescence, and readings obtained with the highest supplement concentrations were equated with 100% proliferation. The experiments were set up in duplicate and repeated three times with similar results.

    Techniques Used: Western Blot, SDS Page, Incubation, Fluorescence

    40) Product Images from "Adenosine A2B and A3 receptor location at the mouse neuromuscular junction"

    Article Title: Adenosine A2B and A3 receptor location at the mouse neuromuscular junction

    Journal: Journal of Anatomy

    doi: 10.1111/joa.12188

    Localization of the A 3 R receptors by immunohistochemistry at the neuromuscular junction. In these adult muscles, A 3 R receptors are strongly immunolabeled in the neuromuscular junction. (A–D) images were obtained using the rabbit polyclonal antibody
    Figure Legend Snippet: Localization of the A 3 R receptors by immunohistochemistry at the neuromuscular junction. In these adult muscles, A 3 R receptors are strongly immunolabeled in the neuromuscular junction. (A–D) images were obtained using the rabbit polyclonal antibody

    Techniques Used: Immunohistochemistry, Immunolabeling

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    Article Snippet: .. H-NS was detected by using polyclonal anti-H-NS antibodies and peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (Sigma) with the aid of a chemiluminescence detection system according to the manufacturer's protocol (Immobilon Western [Millipore]). .. The level of ribosome release factor (RRF), an internal loading control, was detected by using purified anti-RRF antibodies.

    Article Title: The Insulin-Like Growth Factor 1 Receptor Is Essential for Axonal Regeneration in Adult Central Nervous System Neurons
    Article Snippet: .. Primary Antibodies The following primary antibodies were used: mouse monoclonal antibody to c-myc (Sigma) diluted 1∶600; mouse monoclonal antibody to the axonal marker Tau-1 (Calbiochem) diluted 1∶600; rabbit polyclonal antibody to βgc 1∶50 for immunofluorescence and 1∶250 for Western blots; rabbit polyclonal antibody to the neurofilament 200 kD sub-unit (NF200) (Sigma) diluted 1∶600 (NF200 is expressed by RGC and horizontal cells in the retina: ); rabbit monoclonal antibody to Phospho-IGF-1 Receptor (tyr980-C14A11-Cell Signaling) diluted 1∶50; goat polyclonal antibody to IGF-1r β subunit (20C sc-713-G, Santa Cruz Biotechnology) diluted 1∶250 for Western blots and a rabbit monoclonal antibody to phospho p85 (tyr458)/p55(Tyr199) (Cell Signaling) diluted 1∶200. .. Cell Culture Retinal cultures were prepared essentially as previously described , , .

    Chromatin Immunoprecipitation:

    Article Title: Myb-binding Protein 1a (Mybbp1a) Regulates Levels and Processing of Pre-ribosomal RNA *
    Article Snippet: .. The following antibodies were used for Western blotting, ChIP, and immunostaining: rabbit polyclonal α-RPA116 , rabbit polyclonal α-PAF53 , and α-TIF-IA (Ref. ; kindly provided by I. Grummt), rabbit polyclonal α-Mybbp1a (generated to a GST fusion protein containing amino acid residues 976–1263 of human Mybbp1a) and α-mouse Mybbp1a , mouse monoclonal α-FLAG (M2, Sigma), rabbit polyclonal α-FLAG (Sigma), mouse monoclonal α-tubulin (Dm1a, Sigma), rabbit polyclonal α-RPA194 (sc-28174, Santa Cruz Biotechnology), α-BrdU (Roche Applied Science), α-Pes1 , α-TTF-I , α-fibrillarin (P2G3 ( )), α-B23 (sc-56622, Santa Cruz Biotechnology), α-EBP2 (kindly provided by L. Frappier ( )), α-rpS2 (kindly provided by M. Bedford ( )), α-DDX21 (Proteintech Group), α-Nol1, α-GFP-Alexa488 (GBA-488, ChromoTek), goat-α-mouse-Alexa488 F(ab′)2 (Molecular Probes), and goat-α-rabbit-Alexa594 (Molecular Probes). .. HeLa cells were transfected with Polyfect reagent (Qiagen) according to the manufacturer's instructions with the indicated amounts of expression construct together with empty vector DNA to adjust DNA concentration and the pHrD-IRES firefly reporter construct (a kind gift of S. T. Jacob ( )) as a reporter for RNA Pol I transcription activity.

    Immunofluorescence:

    Article Title: The Insulin-Like Growth Factor 1 Receptor Is Essential for Axonal Regeneration in Adult Central Nervous System Neurons
    Article Snippet: .. Primary Antibodies The following primary antibodies were used: mouse monoclonal antibody to c-myc (Sigma) diluted 1∶600; mouse monoclonal antibody to the axonal marker Tau-1 (Calbiochem) diluted 1∶600; rabbit polyclonal antibody to βgc 1∶50 for immunofluorescence and 1∶250 for Western blots; rabbit polyclonal antibody to the neurofilament 200 kD sub-unit (NF200) (Sigma) diluted 1∶600 (NF200 is expressed by RGC and horizontal cells in the retina: ); rabbit monoclonal antibody to Phospho-IGF-1 Receptor (tyr980-C14A11-Cell Signaling) diluted 1∶50; goat polyclonal antibody to IGF-1r β subunit (20C sc-713-G, Santa Cruz Biotechnology) diluted 1∶250 for Western blots and a rabbit monoclonal antibody to phospho p85 (tyr458)/p55(Tyr199) (Cell Signaling) diluted 1∶200. .. Cell Culture Retinal cultures were prepared essentially as previously described , , .

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    Alpha 1 Antichymotrypsin Primary Antibody reacts with histiocytes and histiocytic neoplasms Its major application is defining the presence of Alpha 1 Antichymotrypsin in histiocytes and tumors derived from them In
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    Millipore polyclonal antibodies
    Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with <t>polyclonal</t> antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.
    Polyclonal Antibodies, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 228 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rabbit polyclonal anti e1b 19k
    Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or <t>E1B</t> <t>19K.</t> Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).
    Rabbit Polyclonal Anti E1b 19k, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore polyclonal antibody against dinitrophenylhydrazone dnp residues
    sCLU interactions in RBCs membrane. Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with <t>polyclonal</t> antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) <t>Anti-dinitrophenylhydrazone</t> <t>(DNP)</t> immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.
    Polyclonal Antibody Against Dinitrophenylhydrazone Dnp Residues, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.

    Journal: PLoS Genetics

    Article Title: ITPase deficiency causes a Martsolf-like syndrome with a lethal infantile dilated cardiomyopathy

    doi: 10.1371/journal.pgen.1007605

    Figure Lengend Snippet: Creation, morphology and biochemical analysis of Itpa null mouse embryos. (A) Cartoon representation of the mouse Itpa genomic locus and gene structure with a more detailed diagram of exons 2–4 indicating the position of the guideRNAs used to create the null alleles in the mouse lines to create null embryos. Representative western blots are shown of embryonic tissue demonstrating absence of Itpa protein in samples used as “Itpa null”. ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio). Blotting for Tubulin serves as a loading control and each lane on the blot corresponds to an individual lysate sample. (B) Representative coronal and transverse images through the heart from optical projection tomography (OPT) of wild-type (top panel) and Itpa -null (bottom panel) e16.5 embryos. The bar charts to the right of this image shows quantification of the heart wall to total heart area ratio which showed no difference between null (orange) and control (green) embryos. (C) Oxidative enzyme histochemistry of wild-type and Itpa -null embryonic heart. Sections were subjected to H E staining, individual COX and SDH reactions together with sequential COX/SDH histochemistry. No evidence of morphological changes or focal enzyme deficiency in the Itpa -null heart was identified. Data are representative of duplicate experiments.

    Article Snippet: ITPA protein is detected in lysates from control but not Itpa -null cells upon probing the blot with polyclonal antibodies raised to full-length ITPA (Millipore) and an N-terminal domain of the protein encoded by sequence 5’ of that mutated by CRISPR (LSBio).

    Techniques: Western Blot, Sequencing, CRISPR, Staining

    Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

    Journal: Genes & Development

    Article Title: Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

    doi: 10.1101/gad.1204904

    Figure Lengend Snippet: Formation of tumor giant cells in tumors defective for apoptosis. ( A ) Hematoxylin and eosin-stained sections reveal numerous tumor giant cells in tumors from transformed BMK cells expressing BCL-2 or E1B 19K. Typical sections of carcinomas as described in the text are shown at a magnification of 200× and highlight areas enriched for tumor giant cells in tumors from animals injected with transformed BMK cells expressing BCL-2 or E1B 19K, with insets of grossly polyploid cells in mitosis (at 1000× magnification). Several tumor giant cells in each image are indicated by white arrows. Note the absence of tumor giant cells in tumors formed by the W2.3.1–5 cells. A typical section of a tumor area enriched for tumor giant cells was also immunostained for adenovirus E1A to demonstrate that the tumor giant cells are derived from the input-transformed BMK cells. Note the numerous tumor giant cells that stain brown in the E1A immunohistochemistry, including several examples indicated by arrows. A tumor section (20 μm) enriched for tumor giant cells (arrows) was stained with YOYO-1 to reveal DNA content as described in the text. This image is shown at 630×. ( B ) Aberrant metaphases and polyploid cells accumulate during tumor progression. Tumor sections were developed by immunohistochemistry using antibodies specific for phospho-histone H3 and are shown at 200×. Black arrows indicate aberrant polyploid mitotic arrays, and mitotic arrays presented at 600× in the insets are boxed. Top panels represent images of sections from mature tumors. Phosphohistone H3 immunohistochemistry of sections of transformed BMK cell masses excised from mice on days 2 and 9 after injection are shown in the bottom two rows (200×). Insets in the phospho-histone H3 panels were photographed at 600×, and areas present in the insets are boxed. Grossly aberrant mitotic arrays stained for phospho-histone H3 that are evident in the W2.Bcl2–3 and D3.zeo-2, but not W2.3.1–5, cells on day 9 are indicated in the insets by white arrows. Necrotic centers are indicated (N).

    Article Snippet: Primary antibodies used were hamster anti-human BCL-2 (BD-Pharmingen); rabbit polyclonal anti-E1B 19K , mouse anti-actin (Oncogene Research Products), rabbit anti-mouse HIF-1α (Cayman), rabbit anti-BIM (Axxora), or rabbit polyclonal antisera raised in our lab against a GST-human PUMA fusion protein encoding a 102-amino acid region common to PUMA-α and PUMA-β.

    Techniques: Staining, Transformation Assay, Expressing, Injection, Derivative Assay, Immunohistochemistry, Mouse Assay

    Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

    Journal: Genes & Development

    Article Title: Hypoxia and defective apoptosis drive genomic instability and tumorigenesis

    doi: 10.1101/gad.1204904

    Figure Lengend Snippet: Antiapoptotic BCL-2 family proteins block apoptosis and promote tumor formation. ( A ) Generation of stable cell lines. Cell extracts made from stable BMK cells that express both BAX and BAK (W2), or that are deficient for BAX and BAK (D3), were subjected to Western blotting with antibodies specific for BCL-2 ( left top panel) or E1B 19K ( right top panel). Note the similar expression levels of each exogenous protein in three independent clones (depicted numerically) and undetectable levels of each exogenous protein in the vector-only control cell lines (W2.3.1–2,5,6 or D3.zeo-1,2,3). Blots were then reprobed with an antibody to actin to verify nearly equivalent levels of protein in all lanes, shown below the BCL-2 and E1B 19K panels. ( B ) BCL-2 and E1B 19K block apoptosis in response to staurosporine. Stable BMK cell lines expressing BCL-2, E1B 19K, and controls were treated with media alone (open bars) or media containing 0.4 μM staurosporine (filled bars) for 24 h, and the viable cell number was determined by trypan blue exclusion. Results are presented as the percent of viable cells in each condition, which in each case represents the average of three independent plates. ( C ) BCL-2 and E1B 19K antagonize BAX and BAK to promote tumor formation. Three independent stable BMK cell lines (circles, squares, and diamonds) expressing BCL-2 (green symbols), E1B 19K (blue symbols), or controls (red symbols) were injected subcutaneously into nude mice, and tumor formation was monitored over time. Each point represents the average tumor volume for five injected animals. W2 cells, which express both BAX and BAK, are shown in the left panel. D3 cells, which are deficient for both BAX and BAK, are shown in the right panel. Note that BCL-2 or E1B 19K expression caused a profound acceleration of tumor formation in the W2 cells, whereas the kinetics of tumor formation in the D3 cells, which are deficient for both BAX and BAK, were unchanged by BCL-2 or E1B 19K expression.

    Article Snippet: Primary antibodies used were hamster anti-human BCL-2 (BD-Pharmingen); rabbit polyclonal anti-E1B 19K , mouse anti-actin (Oncogene Research Products), rabbit anti-mouse HIF-1α (Cayman), rabbit anti-BIM (Axxora), or rabbit polyclonal antisera raised in our lab against a GST-human PUMA fusion protein encoding a 102-amino acid region common to PUMA-α and PUMA-β.

    Techniques: Blocking Assay, Stable Transfection, Western Blot, Expressing, Clone Assay, Plasmid Preparation, Injection, Mouse Assay

    sCLU interactions in RBCs membrane. Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with polyclonal antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) Anti-dinitrophenylhydrazone (DNP) immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.

    Journal: PLoS ONE

    Article Title: Apolipoprotein J/Clusterin in Human Erythrocytes Is Involved in the Molecular Process of Defected Material Disposal during Vesiculation

    doi: 10.1371/journal.pone.0026033

    Figure Lengend Snippet: sCLU interactions in RBCs membrane. Purified RBCs membranes from healthy subjects (N = 6) were lysed in NP-40 and lysates were immunoprecipitated (IP) with polyclonal antibodies against sCLU, Band 3, stomatin or normal serum (control). Immunoprecipitates were immunoblotted (IB) under reducing conditions for sCLU (A 1 , upper panel), Band 3 (A 1 , middle panel), CD59 (A 1 , lower panel) and Hb (A 3 ); shown IPs are representatives from two independent experiments. (A 2 ) CLSM co-immunolocalization of the sCLU and Band 3 proteins at the RBCs plasma membrane. Cells were co-stained with anti-Band 3 monoclonal (green; upper panel) and anti-sCLU polyclonal antibodies (red; lower panel). Captured images were merged to reveal co-distribution sites (yellow; lower panel, arrows). Bars, 3 µm. (B) Anti-dinitrophenylhydrazone (DNP) immunoblotting of sCLU, Band 3, and control (IgGs) immunoprecipitates for the detection of co-immunoprecipitated carbonylated proteins (arrows) in 2,4-dinitrophenylhydrazine-modified (OX) or unmodified protein material.

    Article Snippet: The polyclonal antibody against dinitrophenylhydrazone (DNP) residues was obtained from Millipore (Oxyblot® detection kit, S7150, Chemicon, Temecula, CA).

    Techniques: Purification, Immunoprecipitation, Confocal Laser Scanning Microscopy, Staining, Modification