Journal: Thyroid

Article Title: Evidence That Graves' Ophthalmopathy Immunoglobulins Do Not Directly Activate IGF-1 Receptors

doi: 10.1089/thy.2018.0089

Figure Lengend Snippet: Effects of Igs purified from normal volunteers (NV-Igs) and GO patients (GO-Igs) on the levels of pERK1/2 and pAKT in U2OS-TSHR cells. U2OS-TSHR cells were incubated in HBSS (Basal) or stimulated by 100 ng/mL of insulin-like growth factor 1 (IGF-1), 100 mIU/mL of thyrotropin (TSH), 1:5 diluted NV-Igs (NV#), or 1:5 diluted GO-Igs (GOB# or GOM#), as described in the Materials and Methods. GOB and GOM stand for patient samples from Bethesda, MD, and Mainz, Germany, respectively. pERK1/2 levels (gray bars) and pAKT levels (black bars) were plotted as pg/well (M ± SD) in three experiments.

Article Snippet: Total rabbit anti-IGF-1 receptor beta (#3027S) was purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Purification, Incubation

Journal: Thyroid

Article Title: Evidence That Graves' Ophthalmopathy Immunoglobulins Do Not Directly Activate IGF-1 Receptors

doi: 10.1089/thy.2018.0089

Figure Lengend Snippet: Effects of GO-Igs on the levels of pAKT in U2OS cells. U2OS cells were incubated in HBSS or stimulated by 100 ng/mL of IGF-1 or 1:5 diluted GO-Igs (GOB# or GOM#), as described in the Materials and Methods. GOB and GOM stand for patient samples from Bethesda, MD, and Mainz, Germany, respectively. pAKT levels were plotted as %IGF-1 effect after subtracting basal pAKT (M ± SD) in five experiments.

Article Snippet: Total rabbit anti-IGF-1 receptor beta (#3027S) was purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Incubation

Journal: Thyroid

Article Title: Evidence That Graves' Ophthalmopathy Immunoglobulins Do Not Directly Activate IGF-1 Receptors

doi: 10.1089/thy.2018.0089

Figure Lengend Snippet: Effect of knockdown of IGF-1R on GO-Igs stimulation of pAKT formation in U2OS cells. siRNA was used to knockdown IGF-1R levels in U2OS cells, as described in the Materials and Methods. Control cells, transfected with non-targeting siRNA (NT), and cells in which IGF-1R was decreased by transfection with IGF-1R siRNA (IGF-1R) were incubated in HBSS or stimulated by 100 ng/mL of IGF-1, 100 mIU/mL of TSH, or 1:9 diluted GO-Igs (GOB# and GOM#), as described in Materials and Methods. GOB and GOM stand for patient samples from Bethesda, MD, and Mainz, Germany, respectively. (A) mRNA and protein levels of IGF-1R in NT (gray bars) and IGF-1R siRNA-treated cells (black bars). WB, Western blot. (B) pAKT levels in NT cells (gray bars) and pAKT levels in IGF-1R knockdown cells (black bars) were plotted as %IGF-1 effect in NT cells (M ± SD) in three experiments.

Article Snippet: Total rabbit anti-IGF-1 receptor beta (#3027S) was purchased from Cell Signaling Technologies (Danvers, MA).

Techniques: Transfection, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Insulin-Like-Growth-Factor-Binding-Protein-3 (IGFBP-3) Contrasts Melanoma Progression In Vitro and In Vivo

doi: 10.1371/journal.pone.0098641

Figure Lengend Snippet: (A) Phosphorylation state of the indicated markers in primary (WM 793, light grey) and metastatic (Me501, dark grey) human melanoma cells, untreated. (B) WM 793 cells before (light grey) and after (dark grey) treatment with IGFBP-3 (C) Me501 cells before (light grey) and after (dark grey) treatment with IGFBP-3. The assays were performed and quantified as described in the Methods. All determinations were repeated at least three times (D) Western blot analysis of the phosphorylation state of Akt (Ser 473) in Me501 cells untreated or treated with IGFBP-3 for the indicated times. pAKT 473 = phosphor-(Ser473); AKT = total AKT: Tub = tubulin. The gel shown is representative of experiments that were repeated at least three times.

Article Snippet: Depending on the experiment, the membranes were probed with the following antibodies: rabbit polyclonal anti-phospho-Akt (ser 473); anti-total Akt antibodies; anti-IGF-1 receptor β, total; anti-IGF-1 receptor β (pTyr1135) (Cell Signaling); β-Tubulin (Sigma-Aldrich); rabbit monoclonal anti-tyrosinase antibodies (Epitomics); anti-IGF-1 rabbit monoclonal antibodies (Epitomics); anti-IGFBP-3 rabbit polyclonal antibodies (Acris).

Techniques: Western Blot

Journal: Circulation

Article Title: PTP1b Is a Physiologic Regulator of Vascular Endothelial Growth Factor Signaling in Endothelial Cells

doi: 10.1161/CIRCULATIONAHA.114.009683

Figure Lengend Snippet: Increased IGF-1 and VEGF-A signaling in PTP1bECKO cells relative to WT. A, C, and E, IGF1, FGF2, and VEGF-A signaling in wild-type and phosphotyrosine phosphatase 1b (PTPIb)–null endothelial cells. Western blotting of total cell lysates isolated from floxed PTP1b cells treated with control CMV adenovirus or CRE adenovirus for 3 days and then starved overnight. Confluent, serum-starved cells were stimulated for the times indicated with 50 ng/mL of IGF-1, FGF2 or VEGF-A. A, Phosphorylation of the tyrosine residue 1131 of the IGF1 receptor and p44/p42 MAP kinase is increased in PTP1bECKO cells relative to WT after IGF-1 treatment. C, Phosphorylation of p44/p42 MAP kinase is increased in PTP1bECKO cells relative to wild-type after VEGF-A treatment. E, Phosphorylation of p44/p42 MAP kinase is not changed in PTP1bECKO cells relative to wild-type following FGF2 treatment. B, D, and F, Quantification of relative pERK induction by IGF1, FGF2, and VEGF-A in wild-type and PTP1bECKO cells (n=3; SD, *P<0.05).

Article Snippet: Human fibronectin (BD Biosciences#356008), Matrigel Basement Membrane Matrix Growth Factor Reduced (BD#354230), IGF-1(Sigma), VEGF-A 165 (#293-VE R&D systems); Antibodies used included the following: anti-CD31 (BD#55370), anti–VE-cadherin (Santa Cruz#SC6458), antiphospho p44/42 MAP kinase (phospho-ERK, Cell Signaling#9106), anti-p44/42 MAP kinase (total ERK, Cell Signaling#9102), anti-PTP1b (Santa Cruz#sc-1718), anti phospho IGF-1 receptor β (Tyr1131) (CS#3921), anti IGF-1 receptor β (CS#3027).

Techniques: Western Blot, Isolation