Journal:

Article Title: Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells

doi:

Figure Lengend Snippet: (A) Illustration of the heterodimeric type 1 insulin-like growth factor receptor (IGF-1R) indicating subdomains. The cytoplasmic region of the IGF-1R β chain (βwt; residues 931–1337), which contains the tyrosine kinase domain, was expressed inducibly in Schizosaccharomyces pombe. The C-terminus of the full length IGF-1R was tagged with green fluorescent protein (GFP) to generate GFP–IGF-1R. (B) βwt expression and tyrosine kinase activity in S pombe. Schizosaccharomyces pombe cells transformed with pRSP βwt were induced to express βwt by removal of thiamine from the media (thiamine −), or left uninduced (thiamine +). After 24 hours, cultures were harvested, lysates were prepared, and 10 μg of protein was separated by 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis. Western blot analysis was performed with anti-IGF-1Rβ and antiphosphotyrosine antibodies. The βwt protein is indicated at 47 kDa in the induced culture (thiamine −). A range of antiphosphotyrosine reactive bands in the right panel indicates tyrosine phosphorylation of endogenous yeast proteins

Article Snippet: The mutated IGF-1R was then released from pKS upon digestion with SalI and BamHI, blunt ended by incubating with Taq polymerase for 30 minutes at 72°C, then cloned into the pT-Adv vector (Clontech).

Techniques: Expressing, Activity Assay, Transformation Assay, Polyacrylamide Gel Electrophoresis, Western Blot

Journal:

Article Title: Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells

doi:

Figure Lengend Snippet: Coexpression of protein tyrosine phosphatase 1B (PTP1B) with type 1 insulin-like growth factor (IGF-1) stimulated green fluorescent protein–IGF-1 receptor (GFP–IGF-1R). COS7 cells were transiently transfected with pIGF-1R–GFP + pIRES (left panel) or pIGF-1R–GFP + pIRES-PTP1B (right panel) and stimulated with IGF-1 as before. The top panels show GFP–IGF-1R expression (green). Immunofluorescent staining with an anti-PTP1B antibody indicates expression of endogenous PTP1B in the control vector transfected cells (middle left panel: red) and overexpression of PTP1B in cells transfected with pIRES–PTP1B (middle right panel: brighter red fluorescence). Examination of the merged images indicates colocalisation of GFP–IGF-1R with overexpressed PTP1B in a subpopulation of cells (yellow).

Article Snippet: The mutated IGF-1R was then released from pKS upon digestion with SalI and BamHI, blunt ended by incubating with Taq polymerase for 30 minutes at 72°C, then cloned into the pT-Adv vector (Clontech).

Techniques: Transfection, Expressing, Staining, Plasmid Preparation, Over Expression, Fluorescence

Journal:

Article Title: Identification of an IGF-1R kinase regulatory phosphatase using the fission yeast Schizosaccharomyces pombe and a GFP tagged IGF-1R in mammalian cells

doi:

Figure Lengend Snippet: Immunofluorescent detection of type 1 insulin-like growth factor (IGF-1) induced tyrosine phosphorylation in cells transfected with green fluorescent protein –IGF-1 receptor (GFP–IGF-1R). R− cells were transiently transfected with GFP–IGF-1R and 48 hours after transfection cells were stimulated with IGF-1 for 15 minutes, or left untreated. Cells were fixed and stained for phosphotyrosine as described in the materials and methods. Stained cells were examined and photographed under ×40 magnification, using the blue filter to visualise GFP–IGF-1R (green) and the fluorescein isothiocyanate filter to visualise phosphotyrosine staining (red). Untreated cells are shown in the left panel and colocalisation of red (phosphotyrosine) and green (GFP–IGF-1R) is not seen in the merged image. In IGF-1 stimulated cells, shown in the right panel, colocalisation of GFP–IGF-1R with phosphotyrosine staining is observed, and appears yellow in the merged image.

Article Snippet: The mutated IGF-1R was then released from pKS upon digestion with SalI and BamHI, blunt ended by incubating with Taq polymerase for 30 minutes at 72°C, then cloned into the pT-Adv vector (Clontech).

Techniques: Transfection, Staining

Journal: Journal of Biological Chemistry

Article Title: A Self-defeating Anabolic Program Leads to β-Cell Apoptosis in Endoplasmic Reticulum Stress-induced Diabetes via Regulation of Amino Acid Flux

doi: 10.1074/jbc.m113.466920

Figure Lengend Snippet: FIGURE 6. Attenuation of translational recovery during ER stress in Min6 cells promotes survival. A, [35S]Met/Cys incorporation into proteins in cells treated with Tg and Sal003 for the indicated times. Sal003 was added 6 h after initiation of Tg treatment as indicated by the scheme. B, Western blot analysis of the indicated proteins from cells treated with Tg and Sal003 (7.5 M for the last 6 or 12 h of Tg treatment). Antibodies against LRS and XPOT were from Abcam;othersarelistedinFig.1.C,LeuandMeAIBuptakeincellstreatedwith Tg and Sal003 (7.5 M) for the indicated times. D, cells were treated as indi- cated and apoptosis was assessed by measuring the percentage of sub-G1 cells by flow cytometry of propidium iodide-stained cells. Leu starvation was performed by replacing the media after 6 h of Tg treatment with Tg-contain- ing Leu-free medium. D-Leu (40 mM) or Sal003 (7.5 M) was added for the last 6 h of Tg treatment. Significant differences (p 0.01) from untreated cells (*) or the appropriate Tg-treated cells (**) are indicated. Error bars, S.E.

Article Snippet: Sal003 was from Tocris.

Techniques: Western Blot, Flow Cytometry, Staining