Recombinant human IGF-I was purchased from R&D System (Wiesbaden, Germany). The dual IGF-IR/IR inhibitor OSI-906 was purchased from SelleckBio (USA). Specific PI3K/Akt inhibitor LY294002 was purchased from Sigma (St. Louis, MO), and specific ERK1/2 inhibitor PD98059 was purchased from Promega (Madison, WI). Proteasome inhibitor bortezomib (PS-341) was purchased from Millenium Pharmaceuticals Inc (Cambridge, MA, USA). Anti-E-cadherin, anti-Vimentin, anti-ZEB1, anti-IGF-IR, anti-phospho-IGF-IR (Tyr1131), anti-phospho-GSK-3β, anti-GSK-3β and anti-phospho-P53 (Ser15) were purchased from Cell Signaling Technology (Beverly, MA). Anti-Snail and anti-Twist2 were purchased from Abcam (Cambridge, MA). All the other antibodies were purchased from Santa Cruz Biotechnology (USA).
Anti igf ir
Manufactured by Cell Signaling Technology
Sourced in Germany, United States
Anti-IGF-IR is a laboratory reagent that specifically binds to the insulin-like growth factor 1 receptor (IGF-IR). It can be used to detect and quantify the expression of IGF-IR in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (2 mentions)
Recombinant human igf 1, by R&D Systems (1 mentions)
Anti phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Ly294002, by Merck Group (1 mentions)
Anti e cadherin, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Pd98059, by Promega (1 mentions)
Anti zeb1, by Cell Signaling Technology (1 mentions)
Anti phospho gsk3β, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti bcl 2, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Signalfire ecl reagent, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti rb, by Cell Signaling Technology (1 mentions)
Anti pcna, by Cell Signaling Technology (1 mentions)
Molecular imager chemidoc system, by Bio-Rad (1 mentions)
Anti pcreb, by Cell Signaling Technology (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
6 protocols using anti igf ir
1
Investigating IGF-I Signaling Pathways
2
Antibody Immunoblotting for Protein Analysis
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The following primary antibodies were used: anti-Akt (monoclonal rabbit, C67E7, #4691), anti-phospho (p)-Akt (S473, monoclonal rabbit, D9E, #4060), anti-Bcl-xL (monoclonal rabbit, 54H6, #2764), anti-Bcl-2 (polyclonal rabbit, #2876), anti-Cyclin D1 (monoclonal rabbit, 92G2, #2978), anti-CREB (monoclonal rabbit, 48H2, #9197), anti-p-CREB (S133, monoclonal rabbit, 87G3, #9198), anti-GAPDH (monoclonal rabbit, D16H11, #5174), anti-IGF-IR (monoclonal rabbit, D23H3, #9750 and monoclonal rabbit, D4O6W, #14,534), anti-phospho (p)-IGF-IR (Y1135/1136, monoclonal rabbit, 19H7, #3024), anti-PCNA (monoclonal rabbit, D3H8P, #13,110) and anti-Rb (polyclonal rabbit, #9313) (all purchased from Cell Signaling Technology). The SS18-SSX fusion protein was visualized using an anti-SS18/SYT antibody (#8819 Santa Cruz Biotechnology) detecting the N-terminus of SS18 (which is retained in the SS18-SSX fusion oncoprotein). Secondary antibody labeling (Bio-Rad) and immunoblot development were performed using an enhanced chemiluminescence detection kit (SignalFire ECL Reagent; Cell Signaling Technology) and a Molecular Imager ChemiDoc system (Image Lab Software; Bio-Rad), as described previously [14 (link), 40 (link)].
3
Immunoblotting analysis of insulin signaling
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Cell lysates were run on an 8% SDS-PAGE and transferred to Hybond-C nitrocellulose membrane (GE Healthcare, Bucks, UK). Proteins were probed with anti-IR (1:500 Santa Cruz, Heidelberg, Germany) anti-GAPDH or anti-β actin (Millipore, Hertfordshire, UK, both 1:10,000), anti-IR-β (1:750 Santa Cruz, Heidelberg, Germany), anti-p-Akt, anti-Akt, anti-p-MAPK and anti-IGF-IR (all 1:1000), and anti-MAPK (1:500) and anti-p-IGF-IR (1:750) (all from Cell Signalling, Leiden, The Netherlands) with secondary antibodies as follows: anti-rabbit (1:2500; IR, IR-β, p-Akt, Akt, p-MAPK, MAPK, p-IGF-IR, and IGF-IR all at 1 :2000) and anti-mouse (1:10,000 for both GAPDH and β-actin). All antibodies were diluted following the manufacturer’s instructions. Proteins were visualized using supersignal west dura ECL solution (Thermo Fischer, Ulm, Germany) and the UVP ChemiDoc-IT Imaging System (UVP, Bio-Rad, Hertfordshire, UK) and analyzed using Vision Works ls Analysis Software (UVP Inc., Upland, CA, USA).
4
Immunofluorescence Labeling of Cellular Proteins
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Cells were fixed in 4 % sucrose/paraformaldehyde and permeabilized with 0.3 % Triton X-100 in PBS. Neurons were then incubated with 5 % bovine serum albumin (BSA) (Sigma) in PBS + 0.1 % Tween 20, for 1 h at 37 °C, to block nonspecific binding, and incubated with primary antibodies, overnight at 4 °C. Cells were then washed five times with PBS + 0.1 % Tween + 0.5 % BSA and incubated with the appropriate secondary antibodies for 1 h at 37 °C. The coverslips were mounted in a fluorescent mounting medium (DAKO, Denmark) and imaging was performed on a laser scanning Confocal Microscope Leica SP2 AOBS SE, using the 40×/63× oil objective. Primary antibodies used were anti-GFP (1:250, Santa Cruz) and anti-IGF-IR (1:500, Cell Signaling); as secondary antibodies, Alexa Fluor 488 and 594 (1:750, Invitrogen) were employed. The fluorescent dye Hoechst 33342 (0.5 μg/ml, 10″ room temperature) was used to stain nuclei.
5
Dose-Response and Time-Course Analysis of IGF-I Signaling
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For dose-response analysis, subconfluent cells were incubated in serum-deprived medium for 24 h, stimulated with increasing ligand concentrations for 24 h at 37°C and solubilized in radioimmune precipitation (RIPA) buffer; for time-course experiments, subconfluent cells were treated with IGF-I for the indicated time points.
To evaluate IGF-I - dependent activation of DDR1, cells were serum-starved for 24 h, and then stimulated with IGF-I (50 nM) for 24 h.
Cell lysates were subjected to western blot analysis, as previously described [49 (link)]. The following antibodies were used: anti-DDR1 (C-20, sc-532, Santa Cruz Biotechnology), anti-phospho(p)-IR/p-IGF-IR (Y1150/Y1151, 3024, Cell Signaling), anti-IGF-IR (9750, Cell Signaling), anti-p-AKT (Ser473, 9271, Cell Signaling), anti-AKT(9272, Cell Signaling), anti-p-ERK1/2 (T202/Y204, 4370, Cell Signaling), anti-ERK1/2 (9102, Cell Signaling), anti-p-p70 (Thr421/Ser424, 9204, Cell Signaling); anti-p70 (C-18, 230 Santa Cruz Biotechnology); anti-α-tubulin (15246, Abcam); anti-actin (Sigma).
To evaluate IGF-I - dependent activation of DDR1, cells were serum-starved for 24 h, and then stimulated with IGF-I (50 nM) for 24 h.
Cell lysates were subjected to western blot analysis, as previously described [49 (link)]. The following antibodies were used: anti-DDR1 (C-20, sc-532, Santa Cruz Biotechnology), anti-phospho(p)-IR/p-IGF-IR (Y1150/Y1151, 3024, Cell Signaling), anti-IGF-IR (9750, Cell Signaling), anti-p-AKT (Ser473, 9271, Cell Signaling), anti-AKT(9272, Cell Signaling), anti-p-ERK1/2 (T202/Y204, 4370, Cell Signaling), anti-ERK1/2 (9102, Cell Signaling), anti-p-p70 (Thr421/Ser424, 9204, Cell Signaling); anti-p70 (C-18, 230 Santa Cruz Biotechnology); anti-α-tubulin (15246, Abcam); anti-actin (Sigma).
6
Western Blot Analysis of IGF-IR and DCN
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For Western blot analysis, aliquots (40 μg) of whole-cell lysates were separated on 10% SDS-polyacrylamide gels, and electrotransferred onto polyvinylidene membranes. The membranes were incubated with anti-IGF-IR (Cell Signaling Technology, MA, USA), anti-phosphorylated IGF-IR (Cell Signaling Technology, MA, USA), anti-DCN (GTX101250, Genetex, Taiwan), and anti-β-actin (BD Biosciences, CA, USA) antibodies, and the signals were obtained by enhanced chemilluminescence (PIERCE, IL, USA).
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