protease inhibitor  (Millipore)

Journal: Genes & Development

Article Title: Poly-ADP ribosylation of PTEN by tankyrases promotes PTEN degradation and tumor growth

doi: 10.1101/gad.251785.114

Figure Lengend Snippet: PTEN is ADP-ribosylated by tankyrases in vitro and in vivo. ( A , B ) PTEN is PARylated in vivo. ( A ) HCT116 cells were lysed with NETN buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PAR antibodies and immunoblotted using the indicated antibodies. ( B ) HCT116 cells were lysed with NETN denaturing buffer containing PARG inhibitor ADP-HPD (5 μM) and protease inhibitor. Lysates were then immunoprecipitated using control or anti-PTEN antibodies followed by Western blotting as indicated. ( C ) Ribosylation of PTEN by TNKS1 and TNKS2 in vitro. Recombinant TNKS1, TNKS2, and PTEN were subjected to in vitro ribosylation assays in the absence or presence of biotin-labeled NAD + . The recombinant proteins were detected by the indicated antibodies, and the ribosylated proteins were determined with anti-biotin antibody. ( D ) The ribosylation of PTEN by TNKS1 is diminished by tankyrase inhibitor XAV939. The recombinant MBP-PTEN and TNKS1 were subjected to an in vitro ribosylation reaction as described above in the absence or presence of the indicated concentrations of XAV939. ( E ) The catalytic activity of TNKS1 is required for PTEN ribosylation. The MBP-PTEN and immunoprecipitated SFB-tagged wild-type or the catalytically inactive mutant of TNKS1 (TNKS1-PD) were subjected to an in vitro ribosylation reaction followed by Western blotting as indicated. ( F ) The tankyrase-binding motif of PTEN is required for its ribosylation by TNKS1. The recombinant MBP-PTEN, MBP-PTEN-AA, and TNKS1 were subjected to an in vitro ribosylation assay and analyzed by Western blotting as indicated. ( G ) Only double knockdown of TNKS1/2 diminishes the ribosylation of PTEN in vivo. HCT116-derived cells with stable knockdown of TNKS1, TNKS2, TNKS1/2, or PTEN were collected and immunoprecipitated using anti-PTEN antibody. The input and immunoprecipitated proteins were analyzed by Western blotting using the indicated antibodies, and the ribosylation of endogenous PTEN was detected by anti-PAR antibody.

Article Snippet: Cycloheximide, MG132, Wiki4, puromycin, G418, and doxycycline were purchased from Sigma-Aldrich; PAPR1/2 inhibitor Olaparib was from LC Laboratories; PARG inhibitor ADP-HPD and JW55 were from Millipore; and XAV939 was from Sigma-Aldrich and Selleckchem.

Techniques: In Vitro, In Vivo, Protease Inhibitor, Immunoprecipitation, Western Blot, Recombinant, Labeling, Activity Assay, Mutagenesis, Binding Assay, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Glutathione S-Transferase Omega 1 Activity Is Sufficient to Suppress Neurodegeneration in a Drosophila Model of Parkinson Disease *

doi: 10.1074/jbc.M111.291179

Figure Lengend Snippet: DmGSTO1 partially restored mitochondrial F 1 F 0 -ATP synthase activity in park 1 mutants. A , in the presence of GSH, recombinant ATP synthase β subunit was glutathionylated by DmGSTO1A in a dose-dependent manner. B , glutathionylated proteins were immunoprecipitated from thorax extracts with an anti-GSH antibody and were immunoblotted with an anti-ATP synthase β antibody. Glutathionylation of endogenous ATP synthase β subunit in park 1 mutants was regulated by the GSH-conjugating catalytic activity of DmGSTO1A but not by DmGSTO1B. The endogenous levels of the glutathionylated form of the ATP synthase β subunit were decreased even more in park 1 /DmGSTO1 null double mutants. Error bars , S.D. The experimental significance was determined by one-way ANOVA (*, p

Article Snippet: Immunoprecipitation and Glutathionylation Assay Thoraces from 3-day-old male flies were homogenized in lysis buffer containing 1× protease inhibitor mixture (Calbiochem-Merck4Biosciences).

Techniques: Activity Assay, Recombinant, Immunoprecipitation

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: (a) is a control group without treatment while (b), (c), and (d) were treated with CaCO 3 /Dox nanocrystal for 24, 48, and 72 hr, respectively.

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques:

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: TUNEL DNA fragmentation assay on MCF-7 cells after treatment with Dox loaded CaCO 3 nanocrystal. (a) is untreated cells showing complete absent of green fluorescence colour while (b), (c), and (d) are treated cells for incubation period of 24, 48, and 72 h, respectively. The cells showed depicting of TUNEL positive stain progressively according to the duration period.

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques: TUNEL Assay, DNA Fragmentation Assay, Fluorescence, Incubation, Staining

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: In vitro cytotoxicity study of MCF-7 cells for incubation periods of 24, 48, and 72 hr with free Dox and the CaCO 3 /Dox nanocrystals. ∗Means with different superscript are statistically significant P

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques: In Vitro, Incubation

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: FTIR spectra of CaCO 3 nanocrystal, CaCO 3 /Dox loaded nanocrystal, and free Dox.

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques:

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: Apoptotic index MCF-7 cells treated with CaCO 3 /Dox nanocrystal. ∗Means with different superscript are statistically significant P

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques:

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: Inverted light microscope images of morphological changes on MCF-7 treated with Dox nanocrystals.

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques: Light Microscopy

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: In vitro cytotoxicity study of MCF-7 cells for incubation periods of 24, 48, and 72 hr with free DOX and the CaCO 3 /Dox nanocrystals. ∗Means with different superscript are statistically significant P

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques: In Vitro, Incubation

Journal: BioMed Research International

Article Title: In Vitro Ultrastructural Changes of MCF-7 for Metastasise Bone Cancer and Induction of Apoptosis via Mitochondrial Cytochrome C Released by CaCO3/Dox Nanocrystals

doi: 10.1155/2014/391869

Figure Lengend Snippet: (a) TEM micrograph of the Dox-loaded rod-shape nanocrystals and (b) FESEM micrograph of CaCO 3 /Dox nanocrystals.

Article Snippet: The cells were seeded at a density of 1 × 106 per dish and incubated overnight at 37°C; then the cells were treated with various concentrations of CaCO3 /Dox loaded nanocrystal and incubated for 24 h; total protein was extracted from the cells using lysis buffer-containing protease inhibitor cocktail set III and phosphatase inhibitor cocktail set I (Calbiochem, EMD Biosciences, San Diego, CA).

Techniques: Transmission Electron Microscopy

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: rEtpE-C-coated beads enter macrophages by a pathway similar to one that mediates E. chaffeensis entry. (A) Latex beads (red) coated with rEtpE-C by anti-EtpE-C labeling (green) under fluorescence microscopy. Scale bar, 1 µm. (B) Fluorescence and phase contrast merged images of rEtpE-C-coated beads incubated with mouse BMDMs. Cells were pretreated with DMSO (solvent control), MDC, genistein, or PI-PLC for 45 min followed by trypsin treatment to remove beads that were not internalized. Scale bar, 10 µm. (C and D) Numbers of internalized rEtpE-C-coated (C) and non-coated (D) beads/cell incubated with mouse BMDMs pretreated with MDC, genistein, or PI-PLC, relative to DMSO treatment (solvent control) set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Labeling, Fluorescence, Microscopy, Incubation, Planar Chromatography, Standard Deviation

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: EtpE-C binds DNase X. (A) Far-Western blotting of renatured rEtpE-C and rECH0825 on a nitrocellulose membrane incubated with THP-1 cell lysate. Native DNase X was detected with anti-DNase X (α-DNase X), and recombinant proteins were detected with anti-histidine-tag (α-His tag). (B) Western blotting of THP-1 cell lysate following affinity pull-down with rEtpE-C bound to Ni-silica matrix. Bound proteins were eluted with imidazole and labeled with α-DNase X or α-His tag. (C) Western blot analysis of E. chaffeensis- infected THP-1 cell lysate immunoprecipitated with anti-EtpE-C (α-EtpE-C) or control IgG. THP-1 cells were incubated with E. chaffeensis for 30 min, followed by lysis, and immunoprecipitated with α-EtpE-C- or control mouse IgG-bound protein A agarose. The precipitates were subjected to Western blotting with α-DNase X. ** DNase X, * mouse IgG heavy chain. (D) Immunofluorescence labeling of rEtpE-C-coated latex beads (red) incubated with RF/6A cells for 1 h with α-DNase X without permeabilization. Note a cluster of beads colocalizes with host cell-surface DNase X. Scale bar, 5 µm. (E) Selected time-lapse images (0 to 6:38 min) of rEtpE-C-coated beads attached to RF/6A cells expressing DNase X-GFP at 4°C, and time 0 min was set upon raising the temperature to 37°C. The white dashed line denotes the RF/6A cell contour. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm (see also Movie S1 ). (F) Line intensity profile analysis of red (rEtpE-C-beads) and green (DNase X-GFP) signal along the length of the line (slanted white line in the image 5E).

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Far Western Blot, Incubation, Recombinant, Western Blot, Labeling, Infection, Immunoprecipitation, Lysis, Immunofluorescence, Expressing, Microscopy

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: Schematic representation of E. chaffeensis binding and entry into mammalian cells. DNase X is enriched in the lipid raft domains of the cell membrane. Extracellular E. chaffeensis uses its surface protein EtpE C-terminal region to make initial contacts with cell surface DNase X that results in further lateral redistribution and local clustering of DNaseX at the sites of bacterial binding. This binding elicits signals that are relayed down-stream and culminated in host cytoskeletal remodeling, filopodial induction and engulfment of the bound bacteria into an early endosome into the host cell. This receptor-mediated endocytosis can be specifically disrupted by genistein, verapamil or MDC. Latex beads coated with rEtpE-C also bind to cell surface DNase X and follows a similar pattern of entry like that of E. chaffeensis .

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Binding Assay

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: EtpE is expressed by E. chaffeensis in HME patients and infected dogs, and immunization with rEtpE-C protects mice against E. chaffeensis challenge. (A) SDS-PAGE analysis and GelCode Blue staining of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane). rEtpE-N was partially cleaved after its expression in E. coli and thus is visualized as multiple bands. (B) Western blot analysis of rEtpE-N (lane 1) and rEtpE-C (lane 2) (5 µg/lane) with HME patient sera (ID: 72088, MRL1-22, MRL1-40) or control human serum (Control), or sera from dogs experimentally infected with E. chaffeensis (ID: CTUALJ, 3918815, 1425) or control dog serum. The relative band intensity for rEtpE-N/rEtpE-C (75 kDa and 34 kDa bands) assessed by densitometry was shown beneath the panels. (C) Dot-plot analysis of E. chaffeensis load of the blood samples from rEtpE-C-immunized and placebo-immunized mice at 5 days after E. chaffeensis challenge. qPCR of E. chaffeensis 16S rDNA normalized to mouse G3PDH DNA. *Significantly different ( P

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Infection, Mouse Assay, SDS Page, Staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: rEtpE-C-coated latex beads bind and enter non-phagocytic host cells. (A) rEtpE-C-coated beads (arrows), but not rEtpE-N, rECH0825, or rGroEL-coated beads, bind and enter HEK293 cells at 1 h pi. Scale bar, 10 µm. (B) Bar graph showing quantitation of similar experiment as (A) by scoring beads in 100 cells. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Quantitation Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Infection, In Vitro, Western Blot, Immunofluorescence, Labeling, Derivative Assay, Fluorescence, Microscopy, Incubation, Immunostaining, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: PLoS Pathogens

Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

doi: 10.1371/journal.ppat.1003666

Figure Lengend Snippet: Internalization of rEtpE-C-coated beads is dependent on DNase X. (A) Immunofluorescence labeling of rEtpE-C-coated or non-coated beads incubated with human macrophages derived from peripheral blood monocytes. At 30 min pi, cells were labeled with α-DNase X without permeabilization. rEtpE-C-coated beads cluster and colocalize with DNase X on the cell surface, but non-coated beads do not. A single z- plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 5 µm (see also suppl. Fig. S7 and suppl. Movie S2 and S3 ). (B) A selected image showing the orthogonal view of macrophage incubated with rEtpE-C-coated (left panel) or non-coated (right panel) beads in (A). The orthogonal view was obtained from the reconstituted 3-D view of serial z -stack images (combined z-section width of 7.2 µm). Scale bar, 5 µm. The fluorescence intensity profiles of green (DNase X) and red (beads) signals were shown. (C) Fluorescence and phase contrast merged images of rEtpE-C-coated and non-coated beads incubated with BMDMs from DNase X −/− and wild-type mice. Cells and beads were incubated for 45 min followed by trypsin treatment to remove non-internalized beads. Scale bar, 10 µm. (D) Numbers of internalized rEtpE-C-coated beads/cell of similar experiment as (C), relative to the number of non-coated beads set as 100. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

Article Snippet: Far-Western blotting, protein affinity pull-down and co-immunoprecipitation Far-Western blotting was performed using 5 µg of rEtpE-C and rECH0825 that were separated by SDS-PAGE, transferred to a nitrocellulose membrane and renatured with serial guanidinium-HCl treatment followed by incubation with THP-1 cell lysate in NP-40 lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% w/v NP-40, supplemented with 1% protease inhibitor cocktail set III [Calbiochem]) as described .

Techniques: Immunofluorescence, Labeling, Incubation, Derivative Assay, Microscopy, Fluorescence, Mouse Assay, Standard Deviation