Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Protein μ1-specific MAbs detect μ1 conformational change in vitro and within infected cells. (a) 35S-labeled ISVPs and ISVP*s were immunoprecipitated with protein A-conjugated magnetic beads alone (none) or beads bound to μ1-specific MAb 10H2, 8H6, 4A3, or 10F6. Viral particles bound to antibody were quantitated by liquid scintillation counting. Error bars indicate standard deviations. (b to e) ISVPs were allowed to bind to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with MAb 10H2 (b), 8H6 (c), 4A3 (d), or 10F6 (e), followed by goat anti-mouse IgG conjugated to Alexa 488. Bars, 10 μm.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: In Vitro, Infection, Labeling, Immunoprecipitation, Magnetic Beads

Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Anticore serum detects changes in particle structure in vitro and within infected cells. (a) 35S-labeled ISVPs, ISVP*s, and cores were immunoprecipitated with protein A-conjugated magnetic beads alone (none) or beads bound to λ2-specific MAb 7F4 (α-λ2) or anticore serum (α-core). Viral particles bound to antibody were quantitated by liquid scintillation counting. Error bars indicate standard deviations. (b) ISVPs were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with anticore serum, followed by goat anti-rabbit IgG conjugated to Alexa 594. (c) Cells were infected as described for panel b, fixed at 2 h p.i. at 37°C, permeabilized, and coimmunostained with anticore serum and 7F4, followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. Arrows in panel c insets indicate punctate spots detected by 7F4 but not anticore serum. Bars, 10 μm.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: In Vitro, Infection, Labeling, Immunoprecipitation, Magnetic Beads

Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Particles resembling cores are generated in infected cells. (a to c) ISVPs were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 2 h p.i. at 37°C. Cells were permeabilized and coimmunostained with anticore serum and μ1-specific MAb 10H2 (a), 4A3 (b), or σ1-specific MAb 5C6 (c), followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. The arrowhead in panel b indicates a punctate spot detected by both anticore serum and 4A3. (d) Intracellular localization of particles. CV-1 cells were infected as described above and fixed at 2 h p.i. Cells were permeabilized and coimmunostained with anticore serum and anti-LAMP-2 MAb H4B4 (α-LAMP-2), followed by goat anti-rabbit IgG conjugated to Alexa 488 and goat anti-mouse IgG conjugated to Alexa 594. Bars, 10 μm. (e and f) Biochemical evidence for particles resembling cores in infected cells. (e) 35S-labeled ISVPs (∼5 × 105 particles/cell) were allowed to adsorb to CHX-pretreated Mv1Lu cells (∼2 × 106) in 60-mm dishes at 4°C, and cells were harvested and lysed in IP buffer at 0 or 2 h p.i. at 37°C. Cytoplasmic lysates were immunoprecipitated with λ2-specific MAb 7F4 (α-λ2) or anticore serum (α-core). Proteins within the immunoprecipitates were resolved by SDS-PAGE, and viral capsid proteins were detected by phosphorimaging. A reference lane for ISVPs is also shown. Positions of viral proteins are indicated to the left and right. (f) Bands in each lane in panel e corresponding to the μ1 proteolytic fragment δ and core proteins λ1, λ2, and λ3 were quantitated by densitometry as described previously (14). Protein μ1 content within each lane was computed as a ratio of band volumes [δ/(λ1 + λ2 + λ3)] and is shown as a percentage of that obtained for the ISVP reference lane. A similar quantitative analysis was also carried out with cytoplasmic lysates prior to immunoprecipitation (no IP). Averages and standard deviations for two (α-λ2, 0 h) or three lanes from two independent experiments are shown.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: Generated, Infection, Labeling, Immunoprecipitation, SDS Page

Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Kinetics of changes in particle structure and onset of viral protein synthesis within infected cells. ISVPs were allowed to adsorb to untreated or CHX-pretreated Mv1Lu cells in suspension at 4°C, and cells were fixed and permeabilized at different times p.i. at 37°C. CHX-treated cells were immunostained with μ1-specific MAb 10H2, 4A3, or 10F6 or with anticore serum (α-core), followed by goat anti-mouse or goat anti-rabbit IgG conjugated to Alexa 488. Both untreated and CHX-treated cells were immunostained with anti-μNS serum (α-μNS), followed by goat anti-rabbit IgG conjugated to Alexa 488. Cells were analyzed by flow cytometry. (a) Representative histograms. Ctrl. Ab, isotype-matched control antibody. Numbers above each histogram indicate the time in minutes p.i. (b) The geometric mean of each histogram was used as a measure of antibody binding, after subtraction of the value obtained with control antibody. Averages from two independent experiments are shown.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: Infection, Flow Cytometry, Binding Assay

Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Induction of μ1 conformational change and generation of particles resembling cores within cells infected with pr-cores+σ1(μ1-WT) or pr-cores+σ1(μ1-HS). (a to c) pr-cores+σ1(μ1-WT) or pr-cores+σ1(μ1-HS) were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 0 or 2 h p.i. at 37°C. Cells were permeabilized and immunostained with μ1-specific MAb 10H2 (a) or 10F6 (b) or with anticore serum (α-core) (c), followed by goat anti-mouse IgG or goat anti-rabbit IgG conjugated to Alexa 488. Bars, 10 μm. (d and e) Suspension cultures of CHX-pretreated Mv1Lu cells were infected and immunostained as described above and analyzed by flow cytometry. The geometric mean of each histogram was used as a measure of antibody binding, after subtraction of the value obtained with control antibody. Averages and standard deviations from three independent experiments are shown.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: Infection, Flow Cytometry, Binding Assay

Journal:

Article Title: The ? Region of Outer-Capsid Protein ?1 Undergoes Conformational Change and Release from Reovirus Particles during Cell Entry

doi: 10.1128/JVI.77.24.13361-13375.2003

Figure Lengend Snippet: Localization of viral particles, the δ region of μ1, and LAMP-1-positive vacuoles in cells infected with pr-core+σ1(μ1-WT) or pr-core+σ1(μ1-HS). pr-cores+σ1(μ1-WT) (a and c) or pr-cores+σ1(μ1-HS) (b and d) were allowed to adsorb to CHX-pretreated Mv1Lu cells at 4°C, and cells were fixed at 2 h p.i. at 37°C. (a and b) Cells were permeabilized and immunostained with λ2-specific MAb 7F4 (α-λ2), followed by goat anti-mouse IgG conjugated to Alexa 594 and then by μ1-specific MAb 10H2 directly conjugated to Alexa 488. The arrow in the panel a inset indicates a punctate spot detected by 7F4 but not by 10H2. The arrowhead indicates a punctate spot detected by both 7F4 and 10H2. (c and d) Cells were permeabilized and coimmunostained with a LAMP-1-specific polyclonal antiserum (α-LAMP-1) and 10H2, followed by goat anti-mouse IgG conjugated to Alexa 488 and goat anti-rabbit IgG conjugated to Alexa 594. Arrowheads in panel d indicate punctate spots that were detected by 10H2 and that colocalized with LAMP-1-positive structures. Images were captured by spinning-disk confocal microscopy and processed as described in Materials and Methods. Bars, 20 μm.

Article Snippet: In most experiments, cells were preincubated with cycloheximide (CHX) (100 μg/ml) (Sigma-Aldrich) in DMEM for 1 h at 37°C to block protein synthesis.

Techniques: Infection, Confocal Microscopy

Journal: PLoS ONE

Article Title: TGF-β/Smad2/3 Signaling Directly Regulates Several miRNAs in Mouse ES Cells and Early Embryos

doi: 10.1371/journal.pone.0055186

Figure Lengend Snippet: ( A ) Western blot analysis of p-Smad2 and PCNA (loading control) levels in TAG1 ES cells pre-treated with SB-431542+ doxycycline (SB+Dox) for 16 hrs, followed by cycloheximide (CHX) treatment for 2, 4, and 6 hrs or CHX+SB for 2 and 4 hrs. The graph presents relative p-Smad2 levels normalized to corresponding PCNA levels. ( B ) qPCR analysis of pri-miRNA expression in CHX (left side) and CHX+SB (right side) treated TAG1 cells. Asterisks denote statistical comparisons of 2 vs. 0 hr time points (Student’s t-test, **p<0.01, *p<0.05). Pri-miRNA levels were normalized to Gapdh . Pri-miR-7a-2 was not detected. Average of 3 biological replicates (n = 3) performed in triplicates. Error bars show ±SEM.

Article Snippet: Cells were pre-treated with 60 μM SB and 1.5 μg/μl Dox for 16 hrs after which media was replaced with DMEM containing 100 μg/μl cycloheximide (CHX) (Calbiochem, UK) for 2, 4 or 6 hrs.

Techniques: Western Blot, Control, Expressing

Journal: Molecular cancer therapeutics

Article Title: REST is a Novel Prognostic Factor and Therapeutic Target for Medulloblastoma

doi: 10.1158/1535-7163.MCT-11-0990

Figure Lengend Snippet: (A) Q-RT-PCR analysis were performed to measure REST gene expression changes in DAOY and D283 cells in response to HDACI treatment. Normalization and statistical calculations were carried out as described in Figure 3. (B) ChIP analyses were done in HDACI-treated DAOY cells to assess changes in acetylation of histones H3 and H4 within a 2 kb region of the REST promoter. Normalization and statistical calculations were carried out as described in Figure 3. Western blot analysis were done to measure REST levels in DAOY cells treated with (C) various HDACIs (D) with SAHA (5 μM) in the presence or absence of cycloheximide (CHX) (10 mg/ml) for 0-120 minutes (mins) (E) with SAHA (5 μM) in the presence or absence of MG132 (20 μM) for 4 hours (hrs). Actin or GAPDH were used as loading controls.

Article Snippet: Tumor cells were treated with 2.5 μM MS-275 (Alexis, San Diego, CA), 33 nM TSA (Millipore, Billercia, CA), 1.5 mM VPA (Sigma, St Louis, MO), 5 μM SAHA (Caymen Chemicals, Ann Arbor, MI), 100 μg/ml CHX (Sigma, St Louis, MO) or 20 μM MG132 (Calbiochem, La Jolla, CA) for various time periods and processed as outlined below.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Western Blot

Journal: Molecular Cancer

Article Title: Circular RNA hsa_circ_0000467 promotes colorectal cancer progression by promoting eIF4A3-mediated c-Myc translation

doi: 10.1186/s12943-024-02052-5

Figure Lengend Snippet: Circ467 may increase the translation efficiency of c-Myc. A The expression of circ467 and c-Myc in SW480 and HCT116 cells after circ467 overexpression was assessed using RT‒qPCR. B The expression of circ467 and c-Myc in SW480 and HCT116 cells after circ467 knockdown was assessed using RT‒qPCR. C The expression of c-Myc in SW480 and HCT116 cells after circ467 overexpression and cycloheximide (CHX) treatment was assessed by western blotting. D Quantification of c-Myc protein degradation according to C. E , F . Polysomes in cytoplasmic extracts from HCT116 cells after circ467 overexpression or knockdown were fractionated by sucrose gradients, and the relative c-Myc mRNA expression level in the gradient fractions was analyzed using RT‒qPCR. G Diagram showing the interaction between circ467 and c-Myc mRNA. H The interaction between circ467 and c-Myc mRNA in SW480 and HCT116 cells was evaluated by RNA pull-down using an anti-circ467 probe, followed by RT‒qPCR analysis of c-Myc expression. I Schematic diagram of the construction of the wild-type and mutant c-Myc 3’ UTR reporters. J c-Myc reporter activity in SW480 and HCT116 cells cotransfected with the circ467 plasmid and potential c-Myc reporters (PGL3-c-Myc-wt or PGL3-c-Myc-mut) was assessed using a dual luciferase reporter assay. The data shown are representative images or are expressed as the mean ± SD of each group from three separate experiments (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 vs. control, Student’s t test)

Article Snippet: HCT116 cells after circ467 overexpression or knockdown were treated with 100 μg/mL cycloheximide (CHX, Selleck) for 15 min, then the cells were harvested and lysed with hypotonic lysis buffer (5 mM Tris-HCl, 2.5 mM MgCl 2 , 1.5 mM KCl, 2 mM DTT, 0.5% Triton X-100, 0.5% sodium deoxycholate).

Techniques: Expressing, Over Expression, Knockdown, Western Blot, Mutagenesis, Activity Assay, Plasmid Preparation, Luciferase, Reporter Assay, Control