Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 1. A genetic screen in Cos-7 cells to isolate cDNAs whose overexpression interferes with CME. Cos-7 cells are transfected with a library of partial cDNAs fused to eGFP (1). One day after transfection, cells accumulating the TfR at the cell surface are selected using a monoclonal antibody against the TfR and anti-mouse IgG antibody-coated dishes or Cy5-labeled Tf and FACS (2). Selected cells are lysed and plasmids are recovered by electroporation into E. coli (3). Plasmids from individual colonies are either pooled and subjected to a new round of selection (4) or are separately purified to analyze the effect of overexpressing individual eGFP fusion proteins on the internalization of TR-Tf by fluorescence microscopy (5).

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Over Expression, Transfection, Labeling, Electroporation, Selection, Purification, Fluorescence, Microscopy

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 2. Identification of short peptides whose overexpression inhibits CME by panning with antibody-coated plates. (A) Cos-7 cells transfected with pEGFP-N1 (C), pEGFP-C2-EΔ95/295 (eps15) or the plasmids isolated from the library (numbers) were incubated in the presence of TR-Tf. eGFP-expressing cells exhibiting normal, reduced or no uptake were scored. The graph represents the percentage of eGFP-expressing cells showing no Tf uptake. Bar = 10 µm. (B) Peptide sequences in frame with the eGFP initiation codon encoded by the inserts of the isolated plasmids. Yxx∅ and LL motifs are highlighted.

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Over Expression, Transfection, Isolation, Incubation, Expressing

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 3. Peptide 38 bears a bona fide Yxx∅ endocytosis motif. (A) Cos-7 cells transfected with pEGFP-N1 (C) or the indicated plasmids were assayed for their ability to internalize TR-Tf. The fluorescent signals from the eGFP and TR-Tf were analyzed by fluorescence microscopy using the appropriate filters. The graph represents the percentage of eGFP-expressing cells showing no uptake. Bar = 10 µm. (B) GST or GST fused to peptide 38 was bound to glutathione–Sepharose beads and incubated with a rat brain extract. Proteins bound to the beads were separated by SDS–PAGE and analyzed by immunoblotting using an antibody against adaptin β. Two micrograms of total rat brain protein extract were loaded as total (TE).

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Transfection, Fluorescence, Microscopy, Expressing, Incubation, SDS Page, Western Blot

Journal:

Article Title: An efficient genetic screen in mammalian cultured cells

doi: 10.1093/embo-reports/kvf131

Figure Lengend Snippet: Fig. 4. Isolation of two cDNAs from human brain whose overexpression inhibits CME using FACS. (A) Cos-7 cells transfected with pEGFP-C2 (eGFP) or pEGFP-C2-EΔ95/295 (eGFP-EΔ95/295) were incubated in the presence of Cy5-Tf and analyzed by FACS. The x-axis represents the fluorescence intensity of the Cy5-Tf surface labeling per cell. The y-axis represents the number of transfected cells showing a particular fluorescence intensity. The bar indicates the intensity threshold used to screen the brain library (>2 × 101). (B) Cos-7 cells transfected with pEGFP-C2 (C) or the plasmids isolated from the human brain library (numbers) were incubated in the presence of TR-Tf. eGFP-expressing cells exhibiting normal, reduced or no uptake were scored. The graph represents the percentage of eGFP-expressing cells showing no uptake. (C) Cos-7 cells transfected with plasmids 13 and 24 were incubated with TR-Tf. The fluorescent signals from eGFP and TR-Tf were analyzed by fluorescence microscopy using the appropriate filters. Bar = 10 µm. (D) Scheme showing the structure of the eGFP fusion proteins encoded by plasmids 13 and 24.

Article Snippet: Cos-7 cells were obtained from the DSMZ and cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin and 100 µg/ml streptomycin.

Techniques: Isolation, Over Expression, Transfection, Incubation, Fluorescence, Labeling, Expressing, Microscopy

Journal: Virology

Article Title: Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus.

doi: 10.1006/viro.1998.9425

Figure Lengend Snippet: FIG. 2. (A) Analysis of 59 end-dependent scanning and IRES-dependent reporter protein expression in vivo. CAT (solid bars) and LUC (striped bars) activity levels in lysates of CV-1 cells infected with vSP6 and transfected with DNA from each of the plasmids as indicated. (B) Effect of poliovirus infection on IRES-dependent translation. Catalytic activity of reporter proteins expressed in CV-1 cells infected with vSP6, transfected with pBi59BVDV (BVDV) or pBi59polio (polio), and superinfected with poliovirus strain Mahoney (cross-hatched bars) or mock-infected (bars with dotted pattern).

Article Snippet: Transfections were carried out at 37°C and 5% CO2 for 2 h. After transfection, the medium containing the DNA–lipid complexes was removed, and cells were supplemented with 1 ml of Dulbecco’s minimum essential medium and 10% fetal bovine serum for an additional 16 h. Poliovirus type I strain Mahoney (American Type Culture Collection) was propagated in Cos-7 cells.

Techniques: Expressing, In Vivo, Activity Assay, Infection, Transfection