Igf 1r

Manufactured by Cell Signaling Technology

Sourced in United States, China

IGF-1R is a receptor tyrosine kinase that binds insulin-like growth factor 1 (IGF-1). It is involved in cell growth, proliferation, and survival signaling pathways.

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Anti-IGF-1R (40 citations) P-IGF-1R (27 citations) Phospho-IGF-1R (10 citations) Anti-phospho-IGF-1R (7 citations) Anti-p-IGF-1R (5 citations) Anti-IGF-1R antibody (4 citations) Anti-phospho-IGF-1R (Tyr1135/1136) (2 citations) IGF-1R siRNA (2 citations)

95 protocols using igf 1r

Western Blot Analysis of Protein Extracts

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The SAT, VAT, HPAs, and differentiated HPAs were lysed in tissue/cell lysis/extraction reagent (Thermo Scientific, Waltham, MA, USA) supplemented with 1/100 (v/v) protease inhibitor cocktail (Thermo Scientific, Waltham, MA, USA). The protein concentrations of tissue/cell lysates were determined using the Bradford assay. Protein extracts from tissue (30 μg) or cells (15 μg) were separated on a 10% acrylamide gel and transferred to a nitrocellulose membrane (100 V, 2 h). For tissue lysates, blots were probed for FABP4 (1 : 200, Santa Cruz, Texas, USA), CHOP (1 : 1000, Cell Signaling, MA, USA), insulin growth factor 1-receptor (IGF-1R, 1 : 1000, Cell Signaling, MA, USA), and GAPDH (1 : 1000, Santa Cruz, Texas, USA). For cell lysates, blots were stained for IGF-1R (1 : 1000, Cell Signaling, MA, USA), PPARγ (1 : 1000, Cell Signaling, MA, USA), p-Akt (Ser 473) (1 : 1000 Cell Signaling, Danvers, MA, USA), Akt (1 : 1000, Cell Signaling, Danvers, MA, USA), and GAPDH (1 : 1000, Santa Cruz, Texas, USA). Densitometric analysis of the bands was performed with ImageJ (1.45v, National Institutes of Health, USA) to determine the respective protein expression levels.

Wang Y., Mak J.C., Lee M.Y., Xu A, & Ip M.S. (2018). Low-Frequency Intermittent Hypoxia Promotes Subcutaneous Adipogenic Differentiation. Oxidative Medicine and Cellular Longevity, 2018, 4501757.

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Western blot analysis of AKT and IGF-1R signaling

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Western blot was performed as described earlier 11 (link). In short, total proteins were extracted from U87 and U251 cells using RIPA buffer with protease inhibitors. Protein levels of AKT, p-AKT, IGF-1R and p-IGF-1R then analyzed by SDS-PAGE and transferred for immunoblotting with primary antibodies overnight at 4 °C. Then the membranes were incubated with secondary antibodies for 2 hours. Immunoblots were visualized using ECL chemiluminescent detection system and relative integrated density values (IDVs) were calculated by FLuor Chem2.0 software using GAPDH as a control for protein normalization. The antibodies were listed in the following: IGF-1R, p-IGF-1R, AKT, p-AKT (1:1000; Cell Signaling, Beverly, MA, USA), GAPDH (1:1000, Santa Cruz Biotechnology), Goat anti-rabbit antibody and Goat anti-mouse antibody (1:5000, Proteintech, Hubei, China).

Yu M., Yu S., Gong W., Chen D., Guan J, & Liu Y. (2019). Knockdown of linc01023 restrains glioma proliferation, migration and invasion by regulating IGF-1R/AKT pathway. Journal of Cancer, 10(13), 2961-2968.

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Molecular Response to Taselisib and Letrozole

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Taselisib, also called GDC-0032, was generated at Genentech, Inc. (South San Francisco, CA). Letrozole was obtained from US Biological. Antibodies used include phospho-AKT^Ser473, AKT, phospho-PRAS40^Thr246, phospho-S6^Ser235/236, phospho-S6^Ser240/242, S6, phospho-ERK^{Thr202/Tyr204}, ERK, phospho-ERα^Ser118, phospho-ERα^Ser167, cleaved PARP, p110α, phospho-p70S6K^Thr389, PR, cyclin E, phospho-mTOR^Ser2448, IGF1R, BRCA1, c-Myc, CAV1, HER2 and cyclin D1 obtained from Cell Signaling (Danvers, MA). Antibodies for ERα and ERβ were obtained from Santa Cruz biotechnology (Santa Cruz, CA) and a βActin antibody was obtained from Sigma (St. Louis, MO).

Hoeflich K.P., Guan J., Edgar K.A., O'Brien C., Savage H., Wilson T.R., Neve R.M., Friedman L.S, & Wallin J.J. (2016). The PI3K inhibitor taselisib overcomes letrozole resistance in a breast cancer model expressing aromatase. Genes & Cancer, 7(3-4), 73-85.

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Immunoblotting Analysis of Signaling Pathways

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Cells were lysed in NP40 buffer and analyzed by standard immunoblotting protocols. Antibodies used were directed against: ERK1/2, phospho-ERK1/2, phospho-STAT3, phosphor-Akt S473, IRS1, phospho-IRS1 S636/639, IGF-1R, phospho-S6 ribosomal protein, phosphor-4E-BP1, cyclin D1, cleaved caspase 3 (Cell Signaling, Danvers, MA, USA); β-catenin, SHP2, IRS1, GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA); and α-tubulin (Sigma-Aldrich). Secondary antibodies were from Cell Signaling. Detection was done using Clarity^™ Western ECL Substrate (Biorad, Hercules, CA, USA). Densitometric analysis was perfomed using Quantity One Software (Biorad).

Sanchez-Lopez E., Flashner-Abramson E., Shalapour S., Zhong Z., Taniguchi K., Levitzki A, & Karin M. (2015). Targeting colorectal cancer via its microenvironment by inhibiting IGF-1 Receptor-insulin receptor substrate and STAT3 signaling. Oncogene, 35(20), 2634-2644.

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Immunoblotting Analysis of Signaling Pathways

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Cells were treated with or without C96 for 24 hrs before being applied to lysate preparation in a lysis buffer (50 mM Tris-HCI, pH 7.4, 1% NP-40, 0.5% Na-deoxycholate, and 0.1% SDS, 150 mM NaCl, 2 mM EDTA, 2 mM Na₃VO₄, and 5 mM NaF) [6 (link)]. Equal amounts (30 μg) of total proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) separation, followed by immunoblotting analyses with specific antibodies. Antibodies against PARP, caspase-3, AKT, p-AKT(S473), 4E-BP1, p-4E-BP1(S65), IGF-1R, p-IGF-1R, ERK(p42/p44), and p-ERK(p42/p44) were purchased from Cell Signaling Technologies. Antibodies against mTOR, p-mTOR(S2448), p70S6K, p-p70S6K(S424), and GAPDH were obtained from Abgent (Suzhou, China). Horseradish peroxidase-conjugate secondary Antibodies against mouse or rabbit and GAPDH were purchased from Abgent. All immunoblotting signals were further analyzed with Quality One Quality One software (Bio-Rad, Berkeley, CA, USA).

Tang J., Zhu J., Yu Y., Zhang Z., Chen G., Zhou X., Qiao C., Hou T, & Mao X. (2014). A virtual screen identified C96 as a novel inhibitor of phosphatidylinositol 3-kinase that displays potent preclinical activity against multiple myeloma in vitro and in vivo. Oncotarget, 5(11), 3836-3848.

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Characterization of IGF1R-Deficient R-Cells

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The igf1r deficient R‐cell line was isolated from mouse embryos with a targeted disruption of the igf1r gene by Dr. Renato Baserga's group (Sell et al., 1993). pBABE‐Puro retroviral expression vector (#RTV‐001‐PURO) and Platium‐E packaging cell line (#RV‐101) were bought from Cell Biolabs Inc. (San Diego, CA).
Agar (#214220), BrdU (#550891), 7‐AAD (#559925), mouse anti‐IGF1R (#556000), and FITC labeled mouse anti‐BrdU (#347583) was purchased from Becton, Dickinson and Company (San Jose, CA). Polybrene (sc‐134220), antibodies against GAPDH (sc‐25778), cyclin A (SC‐751), cyclin D (SC‐450), cyclin E (SC‐247), CDK2 (SC‐748), and normal mouse IgG (sc‐2025) were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). IGF‐1R (#3027), pAkt (#9275), pErk (#9101), SUMO‐1 (#4940), CDK4 (#2906), p27 kip1 (#3698), InsR β (#3020), and phospho‐tyrosine (#9411) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Cyclin B1 (ab181593) and CDK1 (ab18) antibodies were provided by Abcam (Cambridge, MA). qRT‐PCR primers for IGF1R (#Hs00609566_m1) and GAPDH (#Mm99999915_g1), puromycin (#A11138), and protein G Dynabeads (#10004D) were provided by Life Technologies (Carlsbad, CA).

Lin Y., Liu H., Waraky A., Haglund F., Agarwal P., Jernberg‐Wiklund H., Warsito D, & Larsson O. (2017). SUMO‐modified insulin‐like growth factor 1 receptor (IGF‐1R) increases cell cycle progression and cell proliferation. Journal of Cellular Physiology, 232(10), 2722-2730.

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Cell Viability and Apoptosis Assays

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CellTiter Glo (Promega) viability assays and clonogenic assays were performed as in reference [13 (link)], using cultures growing in full medium supplemented with 10% FCS. Western blotting was performed as described [13 (link)], using antibodies to: IGF-1R (#3027, Cell Signaling Technology, CST), phospho-Y1135/6 IGF-1R (#3024, CST), phospho-S473 AKT (#4051, CST), total AKT (#9272, CST), phospho-T202/Y204 ERK 1/2 (#4377, CST), total ERK 1/2 (#4695, CST), MGMT (#557045, BD Pharmingen), p53 (#9282, CST) PARP (#9542, CST) and actin (ab8224 or ab8227, Abcam). For each western blot shown, similar results were obtained in 1–2 further independent replicates. Viability and survival data were graphed and curve-fitted (GraphPad Prism v5) to interpolate GI₅₀ and SF₅₀ values (drug concentrations that suppress growth or survival to 50% of control values). Apoptosis was quantified by Apo-ONE^® Homogenous Caspase 3/7 Assay (Promega).

Ramcharan R., Aleksic T., Kamdoum W.P., Gao S., Pfister S.X., Tanner J., Bridges E., Asher R., Watson A.J., Margison G.P., Woodcock M., Repapi E., Li J.L., Middleton M.R, & Macaulay V.M. (2015). IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide. Oncotarget, 6(37), 39877-39890.

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Western Blot Analysis of Apoptosis Markers

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Cells were harvested and lysed with buffer containing 10 mM CHAPS as previously described [8 (link)]. Equal amounts of protein based on total cell number were separated by SDS-PAGE, transferred to PVDF membrane, and incubated with primary antibodies overnight at 4 °C. Secondary HRP-conjugated antibodies against rabbit, mouse, and chicken were then used to visualize the immunoblots via enhanced chemiluminescence (ECL2, Pierce). The following antibodies were used in this study: PARP, GAPDH, cleaved caspase-8, caspase-8, cleaved caspase-9, caspase-9, TRAIL-1/DR4, TRAIL-R2/DR5, IGF-1R, phospho-JNK, and total JNK (Cell Signaling), CIB1 (chicken polyclonal antibody, [9 (link)]), vinculin (Sigma), Alix (Biolegend).

Chung A.H., Leisner T.M., Dardis G.J., Bivins M.M., Keller A.L, & Parise L.V. (2019). CIB1 depletion with docetaxel or TRAIL enhances triple-negative breast cancer cell death. Cancer Cell International, 19, 26.

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Western Blot Analysis of Insulin Signaling Proteins

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Cells were lysed with lysis buffer containing 20 mM Tris–HCl (pH 7.4), 5 mM EDTA, 1% NP-40, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, and a protease inhibitor cocktail (#862209, ThermoFisher Scientific, MA, USA). Whole-cell lysates (10 µg) were subject to SDS-PAGE and blotted with specific antibodies. Antibodies against peroxisome proliferator-activated receptor γ (PPARγ) (sc-7273, Santa Cruz Biotech, Santa Cruz, CA, USA), CCAAT/enhancer-binding protein α (C/EBPα) (sc-61, Santa Cruz Biotech), γ-tubulin (T6557, Sigma-Aldrich), pIR (#3023, Cell signaling Technology, Beverly, MA, USA), IR (#3025, Cell signaling Technology), p IGF1R (ab39398, Abcam, Cambridge, MA, USA), IGF1R (#9750, Cell signaling Technology), p IRS1 (I2658-1VL, Sigma-Aldrich), IRS1 (#3407, Cell signaling Technology), pAkt (#4060, Cell signaling Technology), Akt (sc-8312, Santa Cruz Biotech), pERK (#4370, Cell signaling Technology), ERK (#4696, Cell signaling Technology), pGR (#4161, Cell signaling Technology), GR (#12041, Cell signaling Technology), pCREB (#9198, Cell signaling Technology), and CREB (#9197, Cell signaling Technology) were used. Blots were developed using Clarity™ Western ECL Blotting Substrates (BioRad) or ECL-Prime (ThermoFisher Scientific) with a ChemiDoc Imaging System (BioRad).

Lee K.W., An Y.J., Lee J., Lee J.H, & Yim H.S. (2021). α-Poly-l-lysine functions as an adipogenic inducer in 3T3-L1 preadipocytes. Amino Acids, 53(4), 587-596.

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Serum Starvation and Kinase Signaling

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Cells were serum starved for 16 hours in Roswell Park Memorial Institute medium (RPMI) with 0.7% fetal calf serum (FCS) before inhibitor treatment. Cell lysates in RIPA buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxylate, 1% nonyl phenoxypolyethoxylethanol [NP40]) were subjected to western blotting with the following antibodies, all from Cell Signaling Technologies (Danvers, MA, USA): pIGF-1R (3021) (phosphorylated insulin like growth factor-1-receptor); pJAK2 (3771) (phosphorylated janus kinase 2); p STAT5 (9351) (phosphorylated signal transducer and activator of transcription 5); pMEK (9121) (phosphorylated MAPK/ERK kinase); pERK (4370) (phosphorylated extracellular signal regulated kinase); pAKT (9275) (phosphorylated protein kinase B); IGF-1R (3027); JAK2 (3230); STAT5 (9352); MEK (9122); ERK (9102); AKT (9272). Blots were stripped and reprobed against beta actin (Sigma A5441) to ensure equal loading. Immune complexes were detected using chemiluminescence (K12-045-D20, Advansta, San Jose, CA, USA).

Basu T., Bertrand H., Karantzelis N., Gründer A, & Pahl H.L. (2021). Pharmacological Inhibition of Insulin Growth Factor-1 Receptor (IGF-1R) Alone or in Combination With Ruxolitinib Shows Therapeutic Efficacy in Preclinical Myeloproliferative Neoplasm Models. HemaSphere, 5(5), e565.

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