rabbit polyclonal antibody against igf 1r  (Cell Signaling Technology Inc)

Journal: Aging Cell

Article Title: Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions: a study on postmenopausal monozygotic twin pairs

doi: 10.1111/acel.12245

Figure Lengend Snippet: The effects of estrogen stimulation on the expression of miR-182, miR-223, and miR-142-3p and their targets in infant female quadriceps femoris -derived myoblasts. (A) Quantitative PCR analyses of miRNA transcripts normalized with RNU44 in human myoblasts treated for 72 h with 100 n m estradiol or mock. Note: The expression of miR-182 in these myoblasts was too low to be accurately measured. (B) qPCR analyses of target mRNAs (IGF-1, FOXO3A, FOXO1A) in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (C) Representative Western blots of target proteins, IGF-1R, FOXO3A and FOXO1A, and GAPDH, in human myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (D) Densitometry data of Western blots normalized with GAPDH. (E) Representative Western blots showing phosphorylation of AKT and mTOR proteins in myoblasts treated with 10 n m and 100 n m estradiol or mock for 72 h. (F) Densitometry data of Western blots normalized with GAPDH. Data are presented as percentage of control (mock) and reported as means ± SD of three independent experiments. OD indicates optical density. t -test, *** P < 0.001 , ** P < 0.01, * P < 0.05.

Article Snippet: For the MCF-7 cell and mouse muscle experiments, membranes were blocked in TBS with 0.1% Tween-20 (TBS-T) containing 5% fat-free dry milk for 60 min and then incubated overnight at 4 °C with rabbit polyclonal primary antibodies against FOXO1A, FOXO3A, IGF-1R, p-AKT, and total AKT diluted 1:1000 (Cell Signalling Technology) and against β-actin diluted 1:10000 (Santa Cruz, CA), which was used to check the uniformity of blotting.

Techniques: Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Control

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: Gluco-incretin signaling controls susceptibility to apoptosis and IGF-1R expression. A : Islets from control or GipR −/− ;Glp-1R −/− mice were exposed or not to cytokines (IL-1β, TNF-α, and IFN-γ) at low or high concentrations (5, 12.5, and 5 ng/ml or 10, 25, and 10 ng/ml, respectively) and in the presence or absence of GLP-1 (100 nmol/l). B : Quantitative RT-PCR analysis of IGF-1R mRNA expression in islets from control (wt) and GipR −/− ;Glp-1R −/− mice (dKO). C : Reduced expression of IGF-1R expression in GipR −/− ;Glp-1R −/− mouse islets as detected by Western blot analysis. Lower panel : Quantitation of the expression data. D : GLP-1 (100 nmol/l) treatment for 18 h increased IGF-1R expression as detected by Western blot analysis. Lower panel : Quantitation of the expression data. For all panels, data are means ± SD, n = 3 independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Quantitation Assay

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: GLP-1 increased IGF-1R expression and signaling in MIN6 cells. A : MIN6 cells were treated with GLP-1 for the indicated periods of time, and IGF-1R expression and Akt phosphorylation were analyzed by Western blot analysis. B : MIN6 cells were transfected with a control (Un) or an IGF-1R siRNA and treated for 18 h with GLP-1. GLP-1 induced IGF-1 receptor expression, Akt phosphorylation on both T308 and S473, and Bad phosphorylation. Preventing IGF-1R expression suppressed GLP-1–induced Akt and Bad phosphorylation. For all panels, data are means ± SD, n = 3 independent experiments. *** P < 0.001.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Expressing, Western Blot, Transfection

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: GLP-1–induced Akt phosphorylation depends on IGF-1R but not IR expression. A : MIN6 cells were transfected with control (Un, lanes 3 and 6 ), IGF-1R ( lanes 4 and 7 ), or IR-specific ( lanes 5 and 8 ) siRNAs and exposed (+) or not (−) to GLP-1 for 18 h before Western analysis. Lanes 1 and 2 show the induction by GLP-1 of IGF-1R expression in nontransfected cells. Lane 3 shows the basal level of IGF-1R and IR expression in transfected cells; reducing IGF-1R expression ( lane 4 ) but not the IR ( lane 5 ) reduced Akt phosphorylation. Lane 6 : GLP-1–treated cells showed higher expression of the IGF-1R but not of the IR; reducing IGF-1R expression ( lane 7 ) but not IR ( lane 8 ) reduced Akt phosphorylation. Bottom panel : Quantitation of the data. Data are means ± SD, n = 3 independent experiments. ** P < 0.01; *** P < 0.001. B : MIN6 cells were treated or not for 18 h with GLP-1 to increase IGF-1R expression, then incubated with 2 mmol/l glucose for 2 h, and exposed for 15 min to the indicated concentrations of insulin or IGF-2. IGF-1R and total and phosphorylated Akt were determined by Western blot analysis. C : Quantification of the data in B . * P < 0.05, *** P < 0.001 for IGF-2 vs. insulin; §§ P < 0.01, §§§ P < 0.001 for IGF-2 in control vs. GLP-1–treated cells; ### P < 0.001 for IGF-2 vs. insulin in GLP-1–treated cells. a.u., arbitrary units.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Expressing, Transfection, Western Blot, Quantitation Assay, Incubation

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: Activation of Akt phosphorylation by GLP-1 depends on glucose-induced secretion. A : MIN6 cells were incubated for 18 h with GLP-1 to induce IGF-1R expression, then with 2 mmol/l glucose for 2 h, and finally for 1 h with 2 or 20 mmol/l glucose with or without GLP-1 and nimodipine (1 μmol/l) or diazoxide (200 μmol/l). Bottom panel : Quantitation of the data. B : Pancreatic islets were treated as the MIN6 cells in A. C : qRT-PCR analysis of IGF-1 and IGF-2 mRNA expression in mouse islets and cell sorter–purified β-cells. D : Secretion of IGF-2 by MIN6 cells incubated to 2 or 20 mmol/l glucose and in the presence of nimodipine, diazoxide, or GLP-1, as indicated. Total IGF-2 content was 10 ng/4 × 10 6 cells. A–D : Data are means ± SD, n = 3 independent experiments. ** P < 0.01; *** P < 0.001. a.u., arbitrary units.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Activation Assay, Incubation, Expressing, Quantitation Assay, Quantitative RT-PCR, Purification

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: GLP-1–increased Akt phosphorylation depends on secreted IGF-2. A : MIN6 cells were transfected with an unrelated (Un) or an IGF-2–specific shRNA. Forty-eight hours later, they were stimulated with GLP-1 for 18 h to increase IGF-1R expression and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose. B : MIN6 cells were stimulated with GLP-1 for 18 h and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with nonspecific IgGs or an IGF-2 blocking antibody. C : Pancreatic islets were stimulated with GLP-1 for 18 h and then incubated with 2 mmol/l glucose for 2 h and for 1 h in either 2 or 20 mmol/l glucose and with nonspecific IgGs or an IGF-2 blocking antibody. A–C : Data are means ± SD, n = 3 independent experiments. ** P < 0.01; *** P < 0.001. a.u., arbitrary units.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Transfection, shRNA, Expressing, Incubation, Blocking Assay

Journal: Diabetes

Article Title: Glucagon-Like Peptide-1 Protects β-Cells Against Apoptosis by Increasing the Activity of an Igf-2/Igf-1 Receptor Autocrine Loop

doi: 10.2337/db09-0063

Figure Lengend Snippet: GLP-1–induced protection against cytokine-induced apoptosis depends on activation of an IGF-2/IGF-1R autocrine loop. A : MIN6 cells were transfected with an unrelated or an IGF-1R siRNA, and 48 h later they were incubated in the presence or absence of GLP-1 for 4 h before addition of cytokines (IL-1β, 10 ng/ml; TNF-α, 25 ng/ml; IFN-γ, 10 ng/ml); the incubations were continued for 24 h in the continuous presence of GLP-1. B : MIN6 cells were transfected with an unrelated or an IGF-2 shRNA and then treated as described for A. C : MIN6 cells were incubated in the presence or absence of GLP-1 and in the presence of a nonspecific IgG fraction or an IGF-2 blocking antibody for 4 h before addition of cytokines (see above). D : Islets from β-cell–specific Igf-1R −/− mice or from their control littermates were incubated in the presence or absence of GLP-1 for 4 h before addition of cytokines (see above). E : Control islets were incubated for 4 h in the presence or absence of GLP-1 and with a nonspecific or an IGF-2 blocking antibody before cytokines treatment. F : Islets from GipR −/− ;Glp-1R −/− mice were infected with a control adenovirus (Ad-GFP) or an IGF-1R–expressing adenovirus (Ad-IGF1-R) and 1 day later exposed to cytokines, as described in A. A–F : Data are means ± SD, n = 3 independent experiments. ** P < 0.01; *** P < 0.001.

Article Snippet: Rabbit polyclonal antibody against actin was purchased from Sigma (St. Louis, MO); rabbit polyclonal antibodies against the IGF-1R (C20; sc-713), the insulin receptor (C19; sc-711), and total Akt ([H-136]: sc-8312) were from Santa Cruz Biotechnology (Nunningen, Switzerland); rabbit monoclonal antibody against phosphorylated Akt (pAktThr-308) (no. 4056), rabbit polyclonal antibodies against phosphorylated Akt (pAktSer-473) (no. 9271), phosphorylated Bad (pBadSer-112) (no. 9291), and total Bad (no. 9292) were from Cell Signaling (Danvers, MA); and goat polyclonal antibody against IGF-2 (ab10731) was purchased from Abcam (Cambridge, U.K.).

Techniques: Activation Assay, Transfection, Incubation, shRNA, Blocking Assay, Infection, Expressing

Journal:

Article Title: Targeting GIPC/Synectin in Pancreatic Cancer Inhibits Tumor Growth

doi: 10.1158/1078-0432.CCR-08-2837

Figure Lengend Snippet: (A) Immunoblot against IGF-1R after immunoprecipitation for FLAG (IP:Flag IB:IGF-1R). 48 h before harvesting protein MIAPaCa2 cells were transiently transfected with FLAG tagged wild type GIPC/Synectin. Cells were treated with 100 μM of PSQSSSEA or the control peptide dissolved in DMSO. Proteasome inhibitor was used to sustain IGF-1R expression by inhibiting octapeptide-induced degradation. This immunoblot demonstrated that the blocking peptide is effective in reducing the association between GIPC/Synectin and IGF-1R. The Western Blot for FLAG showed equal transfection efficiency. (B) Western Blot analysis of cells treated with PSQSSSEA (200 μM, 100 μM, control peptide) overnight after incubation with 50ng/ml recombinant human IGF-1 in MIAPaCa2 and AsPC1 pancreatic cancer cells. The analysis showed a down-regulation of IGF-1R. Western blots were scanned and cropped according to the molecular weight of IGF-1R. Beta-actin serves as a loading control.(C) Evaluation of IGF-1R protein levels by immunoblot after treatment with PSQSSSEA and recombinant human IGF-1 in MIAPaCa2 pancreatic cancer cells. Before harvesting of the protein 25μM proteasome inhibitor was added for 2 hours. Immunoblots were scanned and cropped according to the molecular weight of IGF-1R. Beta-actin serves as a loading control.

Article Snippet: Goat polyclonal antibodies against the N-terminus of GIPC (clone N19, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies against the beta-chain of IGF-1R (clone C20, Santa Cruz Biotechnology), and antibodies against β-actin and FLAG (Sigma, St. Louis, MO) were used for immunodetection, followed with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology), and the SuperSignal West Pico substrate (Pierce Biotechnology, Rockford, IL).

Techniques: Western Blot, Immunoprecipitation, Transfection, Expressing, Blocking Assay, Incubation, Recombinant, Molecular Weight

Journal:

Article Title: Targeting GIPC/Synectin in Pancreatic Cancer Inhibits Tumor Growth

doi: 10.1158/1078-0432.CCR-08-2837

Figure Lengend Snippet: (A) Western blot analysis of subcutaneous implanted MIAPaCa2 tumor samples with GIPC knock down and normal GIPC expression. IGF-1R protein levels are reduced in the tumor tissue which expresses the GIPC shRNA. Immunoblots were scanned and cropped according to the molecular weight of IGF-1R and GIPC. (B) Densitometry data of the anti-IGF-1R immunoblot of tissue lysates after peptide and control treatment of SCID mice which grow AsPC1 pancreatic cancer cell subcutaneously. The treated tumors show significantly lower amount of IGF-1R compared to the control (p<0.05 student’s t-test). Analysis was performed with Adobe Photoshop CS2. Western Blots show the IGF-1R protein levels of the AsPC1 tumor tissues with and without peptide treatment. Actin serves as a loading control. Immunblots were scanned and cropped according to the molecular weight of IGF-1R. (C) Densitometry data of an anti-IGF-1R immunblot of MIAPaCa2 tissue lysates after peptide and control treatment. The treated tumors show significantly lower amount of IGF-1R as compared to the controls (p=0.02 student’s t-test). Analysis was performed with Adobe Photoshop CS2. The immunoblot shows IGF-1R protein levels of MIAPaCa2 tumor samples with and without peptide treatment. Actin serves as a loading control. Immunblots were scanned and cropped according to the molecular weight of IGF-1R.

Article Snippet: Goat polyclonal antibodies against the N-terminus of GIPC (clone N19, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit polyclonal antibodies against the beta-chain of IGF-1R (clone C20, Santa Cruz Biotechnology), and antibodies against β-actin and FLAG (Sigma, St. Louis, MO) were used for immunodetection, followed with an HRP-conjugated secondary antibody (Santa Cruz Biotechnology), and the SuperSignal West Pico substrate (Pierce Biotechnology, Rockford, IL).

Techniques: Western Blot, Expressing, shRNA, Molecular Weight