After harvesting, the cells were lysed in protein extraction solution (Intron, Daejeon, Korea). Equal amounts of protein were then loaded and separated on a sodium dodecyl sulfate-polyacrylamide gels, and then the proteins were transferred onto nitrocellulose membranes (Pall Corp., Port Washington, NY, USA). After blocking with 5% skim milk, the membranes were incubated with various primary antibodies. The blots were then incubated with peroxidase-conjugated secondary antibody, and enhanced chemiluminescence (Biomax, Seoul, Korea) was used to visualize the specific proteins. The primary antibodies used in western blot analysis were as follows: actin, sterol regulatory element-binding protein-1 (SREBP-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), SREBP-2, farnesyl-diphosphate farnesyltransferase-1 (FDFT-1), peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1α, mitochondrial transcription factor A (mtTFA), p62, Parkin (Abcam, Cambridge, UK), fatty acid synthase (FASN), peroxisome proliferator-activated receptor-γ (PPAR-γ), insulin-like growth factor-1 receptor (IGF-1R), phospho-IGF-1R, Akt, phospho-Akt, mechanistic target of rapamycin (mTOR), phospho-mTOR, (Cell Signalling Technology, Danvers, MA, USA), stearoyl-coenzyme A desaturase (SCD) (Thermo Scientific, Rockford, IL, USA), and LC-3 (Sigma, St. Louis, MO, USA).
Phospho igf 1r
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-IGF-1R is a primary antibody that detects the phosphorylated form of the insulin-like growth factor 1 receptor (IGF-1R). It is used to study the activation and signaling of the IGF-1R in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (7 mentions)
Phospho erk1 2, by Cell Signaling Technology (6 mentions)
Igf 1r, by Cell Signaling Technology (4 mentions)
Phospho egfr, by Cell Signaling Technology (3 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
Total igf1r, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Pall Corporation (1 mentions)
Srebp1, by Santa Cruz Biotechnology (1 mentions)
Parkin, by Abcam (1 mentions)
Phospho mtor, by Cell Signaling Technology (1 mentions)
Srebp2, by Abcam (1 mentions)
Anti β actin hrp, by Merck Group (1 mentions)
Total erk2, by Santa Cruz Biotechnology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Erk1 2, by Merck Group (1 mentions)
Phospho akt, by Merck Group (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Rsk1 2 3, by Cell Signaling Technology (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
10 protocols using phospho igf 1r
1
Western Blot Analysis of Lipid Metabolism
2
IGF-1 Signaling Pathway Activation
3
Comprehensive Cell Lysis and Immunoblotting
4
Comprehensive Protein Profiling Protocol
5
Protein Expression Analysis Workflow
6
Kinase Phosphorylation Profiling Assay
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Total protein was isolated from cells as indicated in each experiment. The following primary antibodies were used and purchased from Cell Signaling Technology (Beverly, MA): rabbit anti-total EGFR, total ALK, total HER3, total IGF-1R, total ERK1/2, total AKT, phospho-ALK (pTry1604), phospho-EGFR (pTyr1068), phospho-HER3 (pTyr1222), phospho-IGF-1R (pTry1135), phospho-ERK1/2 (pThr202/Tyr204), and phospho-AKT (pSer473). The primary antibodies were diluted 1:1000 in blocking buffer and incubated with the membranes overnight at 4°C. Membranes were washed with Tris-Buffered Saline and Tween 20 (TBST) buffer three times, and goat anti-rabbit IgG secondary antibody was applied for 1 hour at room temperature. HRP-conjugated anti-GAPDH antibody was purchased from Life Technologies and diluted 1:2000 for use.
To detect phosphorylation of human kinases, the Proteome Profiler Human Phospho-Kinase Array Kit from R&D Systems (Minneapolis, MN) was employed. For this assay, 300 μg of total proteins was isolated from cells, and the Phospho-Kinase Array Kit was used according to manufacturer’s instructions to detect the phosphorylation of 43 human kinases.
To detect phosphorylation of human kinases, the Proteome Profiler Human Phospho-Kinase Array Kit from R&D Systems (Minneapolis, MN) was employed. For this assay, 300 μg of total proteins was isolated from cells, and the Phospho-Kinase Array Kit was used according to manufacturer’s instructions to detect the phosphorylation of 43 human kinases.
7
Protein Expression Analysis of Liver Tissues
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Proteins were isolated from liver tissues and analyzed according to the methods described previously. The membranes were probed with antibody against phospho-AMPK, AMPK, phospho-IGF-1R, phospho-Akt, Akt, phospho-Sirt1, Sirt1, phospho-p70S6K, p70S6K (Cell Signaling Technology, Danvers, MA, USA), PPARα, IGFBP-2, IGF-1R, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and then developed using an ECL Western blot detection kit (Amersham Bioscience, Piscataway, NJ, USA).
8
Western Blot Analysis of Signaling
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Cell lysates were prepared and subjected to western blot (WB) analysis as described in detail elsewhere [35 (link)]. Briefly, cells were lysed in LDS sample buffer and analyzed by SDS-PAGE on 4-12% Bis-Tris gels (Invitrogen, Carlsbad, CA, USA). Following electrophoresis, proteins were transferred to nitrocellulose membrane, blocked, and incubated overnight with appropriate primary antibody. Following washing, they were incubated with HRP-labeled secondary antibody from Pierce (Rockford, IL, USA). Detection was performed using enhanced chemiluminescent substrate (Pierce) and exposure to X-ray film or imaged using the Odyssey system (Li-cor, Lincoln, NE, USA). Primary antibodies diluted in 5% BSA in TBST for IGF-1R (1:2,000), phospho-ERK1/2 (1:2,000), total ERK1/2 (1:2,000) phospho-Akt (1:2,000) and phospho-IGF-1R (1:2,000) were from Cell Signaling Technologies (via BioNordika, Stockholm, Sweden). Primary antibodies diluted in 5% non-fat dry milk in TBST for p53 (sc-126, 1:1,000) and GAPDH (sc-25778, 1:4,000) were from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).
9
Signaling Pathway Antibody Analysis
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Antibodies against PARP, BAX, cleaved-caspase 3, cleaved-caspase 9, CDK2, phospho-CDK2, p16, p21, and cyclin D1 were purchased from Proteintech (Chicago, IL, USA). Antibodies specific to IGF-1R, phospho-IGF-1R, JNK, phospho-JNK, p38, phospho-p38, ERK1/2, phospho-ERK1/2, PI3K p85, PI3K p110, AKT, phospho-AKT, GSK3-β, phospho-GSK3-β (Ser9), β-catenin, and phospho-β-catenin were purchased from Cell Signaling (Danvers, MA, USA). Antibodies against Bcl-2, β-actin, and rabbit horseradish peroxidase (HRP)-conjugated antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
10
Western Blot Protein Analysis Protocol
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Proteins were extracted from cells using cell lysis buffer (Cell Signaling Technology, Danvers, Massachusetts, USA) supplemented with proteinase (Roche, Upper Bavaria, Germany) and phosSTOP phosphatase inhibitor cocktail (Roche). Proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes, which were then probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibody, and visualized by ECL (GE Healthcare, Buckinghamshire, UK). Antibodies against phospho-ERK, phospho--Akt, total Akt, phosphor-CRAF, total CRAF, phospho-IGF1R, total IGF1R, phospho-MEK, total MEK, phospho EGFR, Ras, phospho Axl, total B-catenin were purchased from Cell Signaling Technology. Total EGFR and total ERK antibodies were purchased from Santa Cruz Biotechnology (Dallas, Texas, USA) and total Axl was obtained from R&D Systems (Minneapolis, Minnesota, USA). Antibody specific for p-EGFR (1173) was obtained from Novus Biologicals, and antibodies specific for Beta-Tubulin was from Milipore. Anti-Wnt5A antibody was purchased from Abcam (Cambridge, Massachusetts, USA).
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