Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Estimation of buoyant density of 20S proteasomes after sucrose density gradient centrifugation. ( a ) One μg of commercial 20S proteasome was analyzed by sucrose (15–55%) density gradient centrifugation. After 5 h of high-speed centrifugation, eight fractions (F) were collected, pelleted, and analyzed by gradient SDS-PAGE. The gel was stained with ROTI Blue quick (Carl Roth, Karlsruhe, Germany). Tracks: 1–8—gradient fractions (F). The proteasome subunits were detected predominantly in F2 (1076 g/cm 3 ) and significantly less in F3 (1099 g/cm 3 ). Titration of commercial 20S proteasome (Prot.): 300 ng, 150 ng, 75 ng is shown. ( b ) The buoyant density of fractions and density of proteasome subunits (underlined).

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Gradient Centrifugation, Centrifugation, SDS Page, Staining, Titration

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Release of presumed MVs from HEK293T cells expressing EGFP and quantification of fluorescent vesicles. ( a ) Expression of EGFP in HEK293T cells after 16 h in culture. ( b , c ) “Pre-detachment” stage of large MVs. ( d , e ) Floating extracellular vesicles in cell-free supernatant after filtration through the 0.45 µm and ( f ) through the 0.22 µm membranes, respectively. Bar = 100 µm. Arrows indicate fluorescent particles. ( g ) Estimation of the amount of EVs in 1 µL of cell culture media by flow cytometry. Estimations were performed in triplicates ( h ). Sucrose gradient centrifugation of cell supernatant from HEK293T cells. Each fraction was concentrated 250 times. No proteasome subunits were detected in analyzed gradient fractions. Western blotting was performed using mouse anti-20S alpha 1, 2, 3, 5, 6, 7 proteasome subunits. The limit of the detection by Western blot assay was ~1 ng/band.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Expressing, Filtration, Cell Culture, Flow Cytometry, Gradient Centrifugation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Protein pattern of retroviruses after sucrose gradient centrifugation. Estimation of the CA contents. ( a ) Examined fractions with buoyant density 1.15 g/cm 3 –1.17 g/cm 3 . Track 1—HEK293T (negative control); Tracks 2,3—exogenous JSRV load 7 μL; Track 4—pNLAEKA load 7 μL; Track 5-pNLd load 7 μL; Track 6—endogenous FeLV load 1 μL; Track 7—endogenous RD-114 load 1 μL; Track 8-BSA 250 ng; Track 9—BSA 125 ng; Track 10—exogenous MMTV load 5 μL; Track 11—endogenous RD-114 load 1 μL; Track 12—endogenous FeLV load 1 μL; Track 13—endogenous PERV load 7 μL. CA proteins are given in a frame. ( b ) Serial two-fold dilutions of bovine serum albumin (BSA) were used as a reference for the CA of retroviruses. Centrifugation was performed for 2 h at 110,000 rpm. Numbers below the figures ( a , b ) represented relative band intensity estimated using ImageJ software. Viruses (tracks 2, 4, 6, 7, 10, 11, 12, 13) selected for further proteasome analyses are underlined. Gels were stained with ROTI Blue quick (Carl Roth, Karlsruhe, Germany). M+-color gel markers mixed with ECL markers.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Gradient Centrifugation, Negative Control, Centrifugation, Software, Staining

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Proteasome α and β subunits were detected in sucrose gradient fractions of JSRV, PERV, FeLV, RD-114, and recombinant HIV-1 (pNLd and pNLAEKA). ( a ) Nitrocellulose membrane after SDS-PAGE, protein transfer and Ponceau S staining. The membrane contains gradient fractions F1–F5 of the supernatant from HEK293T cells and fractions F1′–F5′ obtained from supernatant of JSRV-producing cells. CA (arrow) indicates JSRV capsid proteins. Numbers above the image indicate fraction numbers. ( b ) Nitrocellulose membrane containing gradient fractions F1–F8 from the supernatant of PERV-producing cells stained with Ponceau S. CA (arrow) indicates PERV capsid proteins. Numbers above the image indicate fraction numbers. Track 9: PERV gradient fraction F3 grown on FCS-free medium. Track 10—gradient fraction F4 obtained from the supernatant of non-infected HEK293T cells (control). Track 11—20S proteasome 3 ng (control). ( c ) Western blot analysis of gradient fractions F1–F5 from HEK293T cells and gradient fractions F1′–F5′ from JSRV-producingcells with antibodies to 20S proteasome α 1, 2, 3, 5, 6, 7 subunits. ( d ) Western blot analysis of proteasome control and gradient fractions obtained from PERV-producing cells with antibodies to 20S proteasome α 1, 2, 3, 5, 6, 7 subunits. ( e ) Serial dilution (from 50 ng to 3 ng) of commercial 20S proteasome (Enzo, Farmingdale, NY, USA) and detection of proteasome alpha subunits in retroviruses from gradients fractions (F4, F5) using anti-20S proteasome α 1, 2, 3, 5, 6, 7 serum. ( f ) Detection of proteasome beta subunits in fractions of gradient purified retroviruses by Western blotting using anti-β1, anti-β2, and anti-β5 antibodies. Isolated viruses were from fractions F4 and F5. The 12 ng of commercial 20S proteasome (Enzo, Farmingdale, NY, USA) was used as the positive control. M+-Size markers mixed with ECL-size markers. Positions of marker proteins are indicated with bars.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Recombinant, Membrane, SDS Page, Staining, Infection, Control, Western Blot, Serial Dilution, Purification, Isolation, Positive Control, Marker

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Analyses of proteasome activity using Me 4 Bodipy-Ahx 3 -L3-VS cell-permeable proteasome activity probe and fluorogenic peptides. ( a ) Samples were incubated for 1 h with the probe and loaded into the 15% Tris-glycine polyacrylamide gel. The gel was analyzed at the excitation wavelength 480 nm and emission wavelength 530 nm ( left panel ). Fifteen microliters of gradient fractions were loaded. Two microliters of diluted FeLV and RD-114 virus preparations were used. The same gel was stained using ROTI Blue quick (Carl Roth, Karlsruhe, Germany) protein stain ( right panel ). M-Prestained Protein Marker II (10–200 kDa) (Servicebio, Wuhan, China). ( b ) Chymotrypsin-like and caspase-like proteasome activities in viral preparations and gradient fractions. Fractions (F4) of gradient-purified supernatant from non-infected HEK293T cells and HEK293T cells transfected with JSRV, PERV, and pNLAEKA expression clones were used. Chymotrypsin-like and caspase-like proteasome activities were determined using fluorogenic substrates Suc-LLVY-AMC and Z-LLE-AMC, correspondingly. Six microliters of gradient fractions were used. Two microliters of diluted FeLV and one microliter of RD-114 virus preparations were used. Only five microliters of MMTV were used as the amount of sample was limited. Tests were performed in triplicates. The experiments with MMTV were performed in duplicates.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Activity Assay, Incubation, Virus, Staining, Marker, Purification, Infection, Transfection, Expressing, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Putative mechanism of proteasome integration into retroviral particle. Proteasomes might occasionally be integrated into viral particles along with other cellular proteins and nucleic acids.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Retroviral

Journal: International Journal of Molecular Sciences

Article Title: Functional 20S Proteasomes in Retroviruses: Evidence in Favor

doi: 10.3390/ijms252111710

Figure Lengend Snippet: Cellular protein targets for functional 20S proteasome in retroviruses.

Article Snippet: Control reactions with 100 nM of the proteasome inhibitor Bortezomib (Tocris, Bristol, UK) were performed to test nonspecific degradation of substrates.

Techniques: Functional Assay, Coagulation, Activity Assay

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Depletion of Geminin by siRNA induces rereplication. Western blot (A) and phase-contrast microscopic images (B) of HCT116 cells treated with Geminin or control (GL2) siRNA for 48 h. Western blots were probed with antibodies specific for Geminin and Vinculin (loading control). (C) Flow cytometric profiles of HCT116 treated with Geminin siRNA for 48 h. Before harvesting, the cells were pulsed with BrdU for 2 h and processed for combined propidium iodide staining together with cyclin B1– and BrdU-specific antibodies. The black bars in the phase-contrast pictures correspond to 10 μm. (D) Summary of the protocol used in Geminin siRNA–treated U2OS cells to measure DNA rereplication within a single cell cycle. (E) Geminin expression levels in U2OS cells released from nocodazole block and treated with GL2 (control) or Geminin siRNA. (F) Distribution of [ 3 H]thymidine-labeled DNA in the CsCl gradient fractions. U2OS cells were processed according to the protocol described in A. DNA was prepared and subjected to CsCl density gradient centrifugation as described in Materials and methods. Y axes: [ 3 H]thymidine radioactivity in cpm; x axes: number of gradient fraction collected from the bottom. The fractions containing LL (light-light), HL (heavy-light), and HH (heavy-heavy) DNA were determined by a series of control experiments (not depicted).

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Western Blot, Control, Staining, Expressing, Blocking Assay, Labeling, Gradient Centrifugation, Radioactivity

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rescue of Geminin siRNA phenotype in cells stably expressing HA-tagged RNAi mutants of Geminin. U2OS cell lines expressing two different mutant HA-tagged versions of Geminin were generated (Geminin-Δi179 and Geminin-Δi179-2). In the cDNA for Geminin, two residues were changed in the siRNA target sequence in a way that the nucleotide sequence, but not the amino acid sequence, was altered (C). The cell lines were treated with Geminin siRNA for 48 h and analyzed by Western blot with antibodies specific for the indicated proteins (A) and by FACS ® analysis for DNA content (B). Note the decrease in cells with DNA content greater than and equal to 4N.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Stable Transfection, Expressing, Mutagenesis, Generated, Sequencing, Western Blot

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Geminin depletion activates the checkpoint response during S phase. U2OS cells were transfected with siRNA to Geminin or control and synchronized in mitosis by nocodazole treatment for 16 h. The cells were subsequently released into the next cell cycle and retransfected with Geminin or control siRNA. The cells were harvested at the indicated time points and analyzed by Western blot using the indicated antibodies (A), and for DNA content by FACS ® analysis (B). (C) Cells grown on the coverslips were labeled with BrdU for 10 min at each time point, pre-extracted with 0.5% Triton X-100 in cytoskeleton buffer, fixed with PFA, and immunostained for BrdU and chromatin-bound MCM2. (D) Kinetics of DNA damage checkpoint induction and progression through mitosis of Geminin- or control siRNA–treated cells. The cells were plated on poly- d -lysine–coated coverslips, fixed, and stained with phosphohistone H3 and γH2AX antibodies. 70 cells were counted for each time point.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Transfection, Control, Western Blot, Labeling, Staining

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rereplication induced by Geminin depletion is dependent on CDT1 and CDC6. HCT116 cells were treated with control (GL2), Geminin, CDT1, and CDC6 siRNA at the indicated combinations for 48 h. Cells were harvested and analyzed for protein expression (A), DNA content (B), and cell proliferation (D). (C) Phase-contrast microscopic images taken of cells treated for 48 h with the siRNA for the indicated transcripts. Note the rescue of giant nuclei formation formed in Geminin siRNA–treated cells when cotransfected with CDC6 or CDT1 siRNAs. (E) Model for the role of Geminin in controlling origin firing in S phase. Rereplication results in the activation of an ATR-CHK1–dependent checkpoint that results in block of entry into mitosis. Even though rereplication leads to DNA strand breaks (e.g., phosphorylation of H2AX and formation of RAD51 nuclear foci) and subsequent p53 activation, functional p53 does not appear to be involved in either regulation of rereplication or the DNA damage checkpoint response.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Control, Expressing, Activation Assay, Blocking Assay, Phospho-proteomics, Functional Assay

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Rereplication induced by Geminin depletion activates CHK1 and a DNA damage response. (A) Western blot analysis of cellular extracts prepared at the indicated time points from HCT116 cells treated with Geminin or control (GL2) siRNAs. S317CHK1, Y15CDC2, and S15p53 indicate the use of antibodies specifically recognizing the phosphorylated amino acid of the proteins. (B) Rereplicated cells contain H2AX and Rad51 nuclear foci. U2OS cells treated with control (GL2) or Geminin siRNA for 48 h were immunostained with rabbit polyclonal H2AX and RAD51 antibodies. (C) Rereplicated cells contain ssDNA coated by RPA70. HCT116 cells were prelabeled with 10 μM BrdU for 24 h, incubated with Geminin or control (GL2) siRNA for 48 h, fixed with methanol, and immunostained with a BrdU-specific antibody without denaturation of DNA. BrdU foci (green) correspond to the sites of ssDNA breaks. RPA immunostaining is shown in red. (D) Formation of giant nuclei and CHK1 activation in TIG3 human diploid fibroblasts treated with Geminin siRNA. TIG3 cells were transfected twice with GL2 (control) or Geminin siRNA, fixed, and stained 96 h after the first transfection.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Western Blot, Control, Incubation, Immunostaining, Activation Assay, Transfection, Staining

Journal: The Journal of Cell Biology

Article Title: Loss of Geminin induces rereplication in the presence of functional p53

doi: 10.1083/jcb.200403106

Figure Lengend Snippet: Loss of p53 is not required for rereplication or DNA damage response. (A) U2OS cells were infected with virus generated by pRetroSuper (pRS) p53 producing short hairpin RNA against p53 or with empty virus. Cells were subsequently treated with Geminin-specific siRNA for 48 h and analyzed by Western blotting and FACS ® analysis. Western blots were probed with antibodies specific for the indicated proteins. Actinomycin D treatment (1 nM) was used as a positive control for p53 activation. (B) HCT116 cells infected with empty virus or E6-expressing virus were treated and analyzed as in A.

Article Snippet: Western blotting was performed according to standard procedures, and proteins were detected using the following antibodies: Geminin (FL-209, Cat. No. 13015; Santa Cruz Biotechnology, Inc.), CDC6 (DCS 181 [ ]), CDT1 (mouse mAb raised against human CDT1), RAD51 (Cat. No. 8349; Santa Cruz Biotechnology, Inc.), and RPA70 (a gift of Jerard Hurwitz, Memorial Sloan-Kettering Cancer Center, New York, NY).

Techniques: Infection, Virus, Generated, shRNA, Western Blot, Positive Control, Activation Assay, Expressing

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 and PKCθ act in the same functional pathway to mediate allograft rejection . We monitored the influence on rejection kinetics of murine skin allografts by recipient mice deficient for various proteins. Grafts were visually scored for signs of rejection using a scale of 1–10, with 1 being completely rejected. An allograft was considered fully rejected when it was >80% necrotic. Graphs represent allograft score ( y axis), with a score of “1” being fully rejected and a score of “10” being fully accepted versus time in days after skin grafting ( x axis). Wild-type BALB/c skin was grafted onto (A) wild-type C57BL/6 mice (BALB/c → BL6), which served as a baseline for graft rejection for subsequent allograft combinations; (B) p50 null mice (BALB/c → p50 null ); (C) PKCθ null mice (BALB/c → PKCθ null ); (D) NOTCH1 conditional knock-out (N1KO) mice (BALB/c → N1KO); (E) wild-type C57BL/6 mice whose NOTCH signaling was abrogated by administering γ-secretase inhibitor (GSI LY411,575; BALB/c → WT + GSI); and (F) PKCθ null mice whose NOTCH signaling was abrogated by administering γ-secretase inhibitor (GSI LY411,575) in chow (BALB/c → PKCθ null + GSI). (G) Day to complete rejection was compared between different groups of recipient mice. For each animal grafted with BALB/c skin, an internal control of C57BL/6 skin was also grafted (BL6 → BL6) to monitor integrity of the grafting technique. Data represent the mean + SEM ( n = 3–16 mice/group). *** P < 0.001; one-way ANOVA with Tukey’s post-test applied.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Functional Assay, Knock-Out, Control

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 associates with PKCθ in stimulated T cells . NOTCH1 and PKCθ co-localize and interact following T cell stimulation. (A) Purified CD4 + T cells from C57BL/6 mice were incubated for 30 min with Dynal beads pre-coated with anti-mouse CD3ε and anti-mouse CD28 on glass slides. Cells were fixed, quenched, permeabilized, blocked, and stained with antibodies to NOTCH1 (green) and PKCθ (red). Proteins were visualized using species-specific, fluorescently conjugated secondary antibodies. *Indicates bead. (B) Jurkat T cells were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for the indicated time periods, and subjected to co-immunoprecipitation with anti-NOTCH1. Immunoprecipitates (upper panel) and 1/100 of input (lower panels) were immunoblotted with anti-PKCθ and anti-NOTCH1. Data are representative of at least three independent experiments.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Cell Stimulation, Purification, Incubation, Staining, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 interacts with CARMA1 and BCL10 following stimulation with anti-CD3ε and anti-CD28 . (A) Jurkat T cells were stimulated with plate-bound anti-human CD3ε and anti-human CD28 and subjected to sucrose gradient centrifugation. All fractions were analyzed by dot blot with anti-cholera toxin-conjugated HRP (upper panel). Pooled fractions were immunoblotted with indicated antibodies (lower panel). (B) Jurkat T cells were stimulated as in (A) for the indicated time periods, followed by co-immunoprecipitation with antibodies specific for NOTCH1, CARMA1, or BCL10. Eluates were subjected to immunoblotting with indicated antibodies. 1/100 of input (shown) was immunoblotted with anti-CARMA1, anti-BCL10, and anti-NOTCH1. Arrow heads beside IB: NOTCH1 represent 120 kDa, transmembrane form (upper arrow head) and 110 kDa intracellular form (lower arrow head) of NOTCH1. Data are representative of at least three independent experiments.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Gradient Centrifugation, Dot Blot, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: Cytosolic N1IC can interact with CARMA1 in a bifluorescence complementation assay . Jurkat T cells were transfected with various constructs of nuclear and/or cytosolic proteins and their physical association was assessed microscopically by their ability to reconstitute two halves of a yellow fluorescent protein reporter. Twenty-four hours after transfection, cells were plated onto glass bottom culture dishes, stimulated with anti-human CD3ε and anti-human CD28, and fluorescent images of live cells were captured using a Zeiss LSM 510 confocal microscope. (A) Nuclear proteins, cFos and cJun, known to interact in the nucleus were co-expressed as a control; (B) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was co-expressed with nuclear cJun; (C) NOTCH1 with an additional nuclear export signal, N1IC–NES, was co-expressed with nuclear cJun; (D) NOTCH1 with an additional nuclear export signal, N1IC–NES was co-expressed with CARMA1, a cytosolic protein; (E) NOTCH1 with an additional nuclear localization signal, N1IC–NLS was CARMA1; (F) NOTCH1 with an additional nuclear export signal, N1IC–NES was expressed alone. Upper panel of all images represent fluorescent channel only; lower panel of all images represent merged fluorescent and DIC images. Data are representative of at least three separate experiments.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Transfection, Construct, Microscopy, Control

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 deficiency prevents the formation of the CBM complex . (A) NOTCH1 expression was efficiently reduced when Jurkat T cells were incubated with viral supernatants. NOTCH1-knock-down Jurkat T cells, or mock-infected control cells, were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for 1 h, harvested for co-immunoprecipitation with indicated antibodies, then subjected to immunoblotting with (B,C) anti-NOTCH1, anti-CARMA1, anti-BCL10, or anti-PKCθ; 1/100 of input was immunoblotted with indicated antibodies, or with (D) antibodies specific for NOTCH1, phosphorylated and total IκBα, phosphorylated and total ERK, and phosphorylated and total p38 MAPK. (E) NOTCH1-knock-down Jurkat T cells, or mock-infected control cells, were stimulated with plate-bound anti-human CD3ε and anti-human CD28 for the times indicated. EMSAs were performed on nuclear extracts using radiolabeled oligonucleotides containing the NF-κB binding sequence. Red filled diamond represents competition with cold probe. Data are representative of at least three independent experiments.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Expressing, Incubation, Knockdown, Infection, Control, Immunoprecipitation, Western Blot, Binding Assay, Sequencing

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: The RAM domain of NOTCH1 mediates its interaction with CARMA1 . (A) GFP–N1IC mutant expression plasmids were transfected into 293T cells and sub-cellular localization of NOTCH1 was examined using fluorescence microscopy. (B) VSV–CARMA1 expression plasmids were transiently co-transfected with GFP empty vector as a control, or with GFP–N1IC mutant plasmids in 293T cells, as indicated. Transfected cells were harvested and cell lysates were co-immunoprecipitated with antibodies specific for GFP. Co-immunoprecipitated proteins were then immunoblotted with anti-VSV or anti-GFP. Ig heavy chain indicates immunoglobulin heavy chain.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Mutagenesis, Expressing, Transfection, Fluorescence, Microscopy, Plasmid Preparation, Control, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: NOTCH1 Can Initiate NF-κB Activation via Cytosolic Interactions with Components of the T Cell Signalosome

doi: 10.3389/fimmu.2014.00249

Figure Lengend Snippet: NOTCH1 increases NF-κB activity through its direct interaction with CARMA1 in the cytoplasm . (A) 293T cells were transiently transfected with GFP-N1IC–NLS or GFP-N1IC–NES and sub-cellular localization was determined using fluorescence microscopy. (B) CARMA1 expression plasmids were transiently co-transfected with GFP empty vector as a control, or with GFP-N1IC–NLS or GFP-N1IC–NES in 293T cells. Cytoplasmic extracts from transfected cells were co-immunoprecipitated with anti-GFP. 1/100 of input (shown) was immunoblotted with anti-VSV and anti-GFP. (C) Vectors expressing cytosolic N1IC–NES, membrane-tethered N1ΔE–PM, and/or cytosolic CARMA1 were co-transfected with an NF-κB luciferase reporter construct into Jurkat T cells, stimulated with PMA and CaI for 3 h, then subjected to a luciferase reporter gene assay. Ig heavy chain indicates immunoglobulin heavy chain. Data are mean + SEM of at least three independent experiments.

Article Snippet: The fixed cells were incubated for 1 h with the primary antibodies against PKC-θ and NOTCH1 (Santa Cruz Biotechnology), washed five times in PSG, and then incubated for 1 h with the fluorochrome-labeled rabbit or donkey-anti-species-specific secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA).

Techniques: Activity Assay, Transfection, Fluorescence, Microscopy, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Membrane, Luciferase, Construct, Reporter Gene Assay