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: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: CoIP-based interaction analysis of HCMV pUL50, EBV BFRF1 and VZV Orf24 with the library clones that exerted shared-hook binding activity in the primary screening of Lib1 with pUL50. 293T cells were transiently transfected with plasmids coding for the HA-tagged groove proteins, pUL50, BFRF1 or Orf24, lacking their TMD, in combination with the Flag-tagged library constructs. The wild-type interaction of truncated pUL53 with pUL50 served as a positive and RFP as a negative control. At three d p.t., cells were lysed and Flag-tagged proteins were immunoprecipitated using mAb-Flag; a chicken Fc fragment served as a specificity control. Lysate controls taken prior to the IP and CoIP samples were subjected to standard Wb analysis using tag-specific antibodies as indicated. Blue numbers: each individual CoIP band was first normalized to the corresponding IP signal and was then set in relation to the reference WT interaction (% intensity) using Aida Image Analyzer v.4.23 software (mean values of triplicate densitometric determinations are given); CoIP was performed once.

Article Snippet: Human embryonic kidney epithelial 293T cells (HEK 293T, CRL-3216, ATCC, Manassas, VA, USA) and HeLa cells (ATCC) were cultivated in Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Clone Assay, Binding Assay, Activity Assay, Transfection, Construct, Negative Control, Immunoprecipitation, Control, Software

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: CoIP-based interaction analysis of HCMV pUL50, EBV BFRF1, VZV Orf24 and HSV-1 pUL34 with the library clones that exerted shared-hook binding activity in the primary screening of Lib1 with BFRF1. 293T cells were transiently transfected with plasmids coding for the HA-tagged groove proteins, pUL50, BFRF1, Orf24, or pUL34, lacking their TMD, in combination with the Flag-tagged library constructs. The wild-type interaction of truncated pUL53 with pUL50 served as a positive and RFP as a negative control. At three d p.t., cells were lysed and Flag-tagged proteins were immunoprecipitated using mAb-Flag; a chicken Fc fragment served as a specificity control. Lysate controls taken prior to the IP and CoIP samples were subjected to standard Wb analysis using tag-specific antibodies as indicated. Blue numbers: each individual CoIP band was first normalized to the corresponding IP signal and was then set in relation to the reference WT interaction (% intensity) using Aida Image Analyzer v.4.23 software (mean values of triplicate densitometric determinations are given); CoIP was performed once.

Article Snippet: Human embryonic kidney epithelial 293T cells (HEK 293T, CRL-3216, ATCC, Manassas, VA, USA) and HeLa cells (ATCC) were cultivated in Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Clone Assay, Binding Assay, Activity Assay, Transfection, Construct, Negative Control, Immunoprecipitation, Control, Software

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: CoIP-based interaction analysis of HCMV pUL50, EBV BFRF1 and VZV Orf24 with the library clones that exerted shared-hook binding activity in the primary screening of Lib2 with BFRF1. 293T cells were transiently transfected with plasmids coding for the HA-tagged groove proteins, pUL50, BFRF1, or Orf24, lacking their TMD, in combination with the Flag-tagged library constructs. The wild-type interaction of truncated pUL53 with pUL50 served as a positive and RFP as a negative control. At three d p.t., cells were lysed and Flag-tagged proteins were immunoprecipitated using mAb-Flag; a chicken Fc fragment served as a specificity control. Lysate controls taken prior to the IP and CoIP samples were subjected to standard Wb analysis using tag-specific antibodies as indicated. Blue numbers: each individual CoIP band was first normalized to the corresponding IP signal and was then set in relation to the reference WT interaction (% intensity) using Aida Image Analyzer v.4.23 software (mean values of triplicate densitometric determinations are given); CoIP was performed once.

Article Snippet: Human embryonic kidney epithelial 293T cells (HEK 293T, CRL-3216, ATCC, Manassas, VA, USA) and HeLa cells (ATCC) were cultivated in Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Clone Assay, Binding Assay, Activity Assay, Transfection, Construct, Negative Control, Immunoprecipitation, Control, Software

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: CoIP-based interaction analysis of sequence-predicted shared-hook constructs with three groove proteins. 293T cells were transiently transfected with constructs coding for the HA-tagged groove proteins, pUL50, BFRF1 or Orf24, either full-length or without TMD, in combination with the Flag-tagged sequence-predicted shared-hook constructs. Positive controls were the original hook proteins pUL53, BFLF2 or Orf27 and RFP served as a negative control. At three d p.t., cells were lysed and HA-tagged proteins were immunoprecipitated using mAb-HA; a chicken Fc fragment served as a specificity control. Lysate controls taken prior to the IP and CoIP samples were subjected to standard Wb analysis using tag-specific antibodies as indicated. Interaction of the sequence-predicted shared-hook constructs is indicated with red symbols. Blue numbers: each individual CoIP band was first normalized to the corresponding IP signal and was then set in relation to the reference WT interaction (% intensity) using Aida Image Analyzer v.4.23 software (mean values of triplicate densitometric determinations are given); CoIP was performed three times.

Article Snippet: Human embryonic kidney epithelial 293T cells (HEK 293T, CRL-3216, ATCC, Manassas, VA, USA) and HeLa cells (ATCC) were cultivated in Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Sequencing, Construct, Transfection, Negative Control, Immunoprecipitation, Control, Software

Journal: Cells

Article Title: ‘Shared-Hook’ and ‘Changed-Hook’ Binding Activities of Herpesviral Core Nuclear Egress Complexes Identified by Random Mutagenesis

doi: 10.3390/cells11244030

Figure Lengend Snippet: Qualitative and quantitative analyses of nuclear rim colocalization patterns: coexpressed sequence-predicted hybrid constructs (HCMV/EBV pUL53 sHook1 and HCMV/VZV pUL53 sHook2) in combination with three different groove proteins (HCMV pUL50, EBV BFRF1 or VZV Orf24). ( A ) HeLa or ( B ) 293T cells were transiently cotransfected with either constructs coding for the Flag-tagged sequence-predicted hooks, sHook1 or sHook2, and the HA-tagged groove proteins pUL50, BFRF1 or Orf24. Two d p.t., cells were fixed, used for immunostaining with tag-specific antibodies and analyzed by confocal imaging. DAPI counterstaining indicated the morphology of nuclei of the respective cells. In both types of cells, the localization patterns of coexpressed construct pairs were analyzed; the scale bar in panels A–C, picture 1 marks 7.5 μm. On a qualitative level of analysis ( A , B ), the yellow arrows indicate complete nuclear rim colocalization, and the white arrows show partial rim colocalization. ( C ) Staining of subcellular localizations of marker proteins in HeLa and 293T cells, i.e., the nuclear lamina, the nucleoplasmic and cytosolic compartments. ( D ) Quantitation of the colocalization patterns of pUL53 sHook1 and sHook2 with groove proteins HCMV pUL50, EBV BFRF1 and VZV Orf24. Localization patterns observed in HeLa and 293T cells were quantified and classified as complete, partial or no colocalization. Mean values ± SD are given as expressed in a percentage of the entire number of counted cells. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Tukey correction; **, p < 0.01; ns, not significant. ( E ) Summarized findings of colocalization obtained for the two sequence-predicted hybrid constructs, combining complete and partial colocalization as total percentages; nd, not determined.

Article Snippet: Human embryonic kidney epithelial 293T cells (HEK 293T, CRL-3216, ATCC, Manassas, VA, USA) and HeLa cells (ATCC) were cultivated in Dulbecco’s modified Eagle medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Sequencing, Construct, Immunostaining, Imaging, Staining, Marker, Quantitation Assay