Journal: Journal of Lipid Research

Article Title: Identification of miR-185 as a regulator of de novo cholesterol biosynthesis and low density lipoprotein uptake

doi: 10.1194/jlr.M041335

Figure Lengend Snippet: miR-185 downregulates SREBP-2 mRNA and protein level by binding to MREs within SREBP-2 mRNA 3′UTR. A: Luciferase activity was quantitated in 293T cells transfected with a control luciferase reporter plasmid (Con Luc), SREBP-2 3′UTR containing reporter plasmid (3′UTR), SREBP-2 3′UTR mutant (3′UTR MUT), and pre-miR-185 (miR185) or control pre-miR (Con miR). Luciferase activity was measured in 293T cells as described in the Materials and Methods section. Bar graphs represent mean ± SEM from three independent experiments. B: The fold change in SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185, Antagamer-miR-185 (Antagomer) (100 nM), or control miR (Con miR) transfected HepG2 cells. Bar graphs represent mean ± SEM from three independent experiments. C: SREBP-2 mRNA was measured by quantitative real-time-PCR in pre-miR-185 or control miR transfected THLE-2 cells. Values were normalized to the level of GAPDH. Bar graphs represent mean ± SEM from three independent experiments. D: SREBP-2 protein level was determined using whole cell lysates by Western analysis in miR-185 overexpressing and control miR transfected HepG2 cells. β-Actin was used as a loading control. FL, full-length SREBP-2; M, mature form of SREBP-2.

Article Snippet: THLE-2, HepG2, and 293T cells were obtained from ATCC.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation, Mutagenesis, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Virology

Article Title: Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

doi: 10.1128/JVI.03117-14

Figure Lengend Snippet: Interaction of PCV2 Cap with dynein intermediate-chain IC1. (A) The lysates of PCV2 and mock-infected PK15 cells were immunoprecipitated with mouse anti-Cap IgG. Immunoblotting was then performed to determine the presence of IC1 and α-tubulin in the Cap immunoprecipitate. (B) Recombinant GST-dCap protein was immobilized on glutathione-Sepharose beads and incubated with recombinant His-IC1, His-TUBA1A, His-TUBA1B, or His-TUBB2A. His-tagged proteins in GST-pulldown assays were examined by immunoblotting with anti-His antibody. The levels of His-tagged proteins were determined by Coomassie blue staining. (C) 293T cells were cotransfected with a myc-tagged dCap expression plasmid together with a flag-IC1 expression plasmid. Only cells expressing myc-dCap or flag-IC1 were included as controls. Cell lysates were immunoprecipitated with an anti-flag antibody or an anti-myc antibody. The resulting precipitates were examined by immunoblotting using an anti-flag or an anti-myc antibody to examine the interaction between myc-dCap and flag-IC1. (D1 and D2) Identification of the IC1 interaction domain on Cap. (D1) Schematic representation of various truncated forms of the PCV2 Cap that were tagged with GST and used to identify the IC1 interaction domain. Constructs are named for each intact domain number, with a hyphen(s) indicating the removed domain(s). (D2) Bacterially expressed His-IC1 was incubated with various truncated forms of Cap tagged with GST and immobilized on glutathione-Sepharose beads. Immunoblotting was then performed to characterize the IC1 interaction domain on Cap. (E) Cells were cotransfected with plasmids expressing dCap (CapΔ1-41) or CapΔ42-100, together with a flag-IC1 expression plasmid. Immunoprecipitation and immunoblotting were then performed to examine the interactions between various forms of Cap and flag-IC1.

Article Snippet: Human embryonic kidney epithelial (HEK) 293T cells (CRL-3216, ATCC) were cultured in Dulbecco modified Eagle medium (DMEM; Gibco).

Techniques: Infection, Immunoprecipitation, Western Blot, Recombinant, Incubation, Staining, Expressing, Plasmid Preparation, Construct

Journal: Journal of Virology

Article Title: Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

doi: 10.1128/JVI.03117-14

Figure Lengend Snippet: Acetylation modifications of α-tubulin and histone 3. (A) 293T cells and HDAC6-overexpressing 293T cells were treated with purified rCap for 6 h, TSA for 3 h, NOC for 3 h, or NaBut for 6 h, and immunoblots of cell lysates was probed with mouse anti-acetylated α-tubulin MAb and rabbit anti-acetylated histone 3 antibody to analyze the level of acetylated α-tubulin and histone 3. Data are represented as means ± standard deviations (SD) (n = 3; one asterisk [*] represents P < 0.05, and two asterisks [**] represent P < 0.01) Ac, acetylated. (B) PK15 cells were infected with PCV2 for 12, 24, 48, and 72 h. Cell lysates were probed with anti-Cap, anti-acetylated histone 3, and anti-histone 3 antibodies in immunoblotting experiments.

Article Snippet: Human embryonic kidney epithelial (HEK) 293T cells (CRL-3216, ATCC) were cultured in Dulbecco modified Eagle medium (DMEM; Gibco).

Techniques: Purification, Western Blot, Infection

Journal: Journal of Virology

Article Title: Circovirus Transport Proceeds via Direct Interaction of the Cytoplasmic Dynein IC1 Subunit with the Viral Capsid Protein

doi: 10.1128/JVI.03117-14

Figure Lengend Snippet: Dynamic colocalization of Cap with dynein IC1 subunit. (A to C) Cells immunostained with pig anti-Cap IgG and mouse anti-IC1 antibody were stained with anti-pig FITC and anti-mouse Alexa 546, respectively. Colocalization of Cap with IC1 was determined at the indicated time points, including in PCV2-infected PK15 cells (A), PCV2-infected 3D4/31 cells (B), and 293T cells expressing exogenous Cap (C). (D) PK15 cells were transfected with the indicated plasmid expressing Cap (Cap/NLS [with NLS]), dCap (Cap/NLS− [without NLS]), or Rep (Rep/NLS [with NLS]). At 24 h posttransfection, cells were examined for the subcellular location of exogenous Cap, dCap, and Rep and of endogenous IC1 with pig anti-Cap IgG, pig anti-Rep pAb, and mouse anti-IC1 antibody, followed by staining with anti-pig FITC and anti-mouse Alexa 546. (E) The samples described for panel A were subjected to separation of subcellular components. The indicated proteins in the nuclear fraction were then tested by immunoblotting to validate intranuclear colocalization of Cap with IC1 in PCV2-infected PK15 cells.

Article Snippet: Human embryonic kidney epithelial (HEK) 293T cells (CRL-3216, ATCC) were cultured in Dulbecco modified Eagle medium (DMEM; Gibco).

Techniques: Staining, Infection, Expressing, Transfection, Plasmid Preparation, Western Blot

Journal:

Article Title: The Long Terminal Repeat of Jaagsiekte Sheep Retrovirus Is Preferentially Active in Differentiated Epithelial Cells of the Lungs

doi:

Figure Lengend Snippet: Cell lines used in this study

Article Snippet: H441 and H358 cells were grown in RPMI 1640 medium (Gibco BRL) adjusted to contain 1.5 g of sodium bicarbonate per liter, 4.5 g of glucose per liter, and 10 mM HEPES with 10% FBS. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Cell line Origin Tissue or cell type ATCC no. or reference A549 Human Lung carcinoma CCL-185 H441 Human BAC HTB-174 H358 Human BAC CRL-5807 293T Human Embryonic kidney Lebkowsky et al. ( 33 ) OA1 Sheep Brain fibroblast CRL-6538 OAT Sheep Sheep testis CRL-6546 FLL Sheep Primary fetal lamb MRI a JS7 Sheep BAC Jassim ( 30 ) CP-MRI Sheep Choroid plexus MRI a CP-ATCC Sheep Choroid plexus CRL-1700 mtCC1-2 Mouse Clara cell Magdaleno et al. ( 34 ) BV2 Mouse Microglia A. Tenner a F9 Mouse Testicular carcinoma CRL-1720 FOP Mouse Mammary carcinoma J. Hassel a IC-21 Mouse Peritoneal macrophage TIB-186 MHS Mouse Alveolar macrophage CRL-2019 C2C12 Mouse Myoblast CRL-1772 ABI-2 Mouse Hybridoma HB-33 MLE-12 Mouse Lung epithelium CRL-2110 TCMK Mouse Mouse kidney CCL-139 NIH 3T3 Mouse Mouse embryo CCL-92 MLE-15 Mouse Type II pneumocyte Wikenheiser et al. ( 59 ) ST3 Mouse Thymus stroma Brightman et al. ( 10 ) Open in a separate window a Cells were provided directly by an investigator or institution with no reference available.

Techniques: