The HCT116 and HT29 cells were lysed in RIPA lysis buffer. Then, equal amounts of protein were resolved by SDS-PAGE analysis and electrotransferred onto a PVDF membrane (Millipore, Schwalbach, Germany), then blocked with 5% skim milk powder and incubated with primary antibody at 4 °C overnight. The primary antibodies used were anti-FAK (#3285, Cell Signaling Technology), anti-IGF1R (#ab39675, Abcam), anti-EGFR (#4267, Cell Signaling Technology), anti-YY1 (#66281-1-Ig, Proteintech), anti-GAPDH (#ab181602, Abcam). Then the membranes were incubated with HRP-conjugated secondary antibody for 1 h at room temperature, the blots were visualized using an enhanced chemiluminescence kit (Pierce, Waltham, MA, USA).
Anti igf1r
Manufactured by Abcam
Sourced in United States
Anti-IGF1R is a mouse monoclonal antibody that recognizes the Insulin-like Growth Factor 1 Receptor (IGF1R). IGF1R is a receptor tyrosine kinase that plays a critical role in cell growth, differentiation, and survival.
Automatically generated - may contain errors
Lab products found in correlation
Anti gapdh, by Abcam (3 mentions)
Anti pten, by Cell Signaling Technology (2 mentions)
Anti p akt ser473, by Cell Signaling Technology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (2 mentions)
Anti fak, by Cell Signaling Technology (1 mentions)
Anti yy1, by Proteintech (1 mentions)
Anti egfr, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Anti nedd4, by Cell Signaling Technology (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Non fat milk, by Merck Group (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Skim milk, by Fujifilm (1 mentions)
Alkaline phosphatase conjugated streptavidin, by Agilent Technologies (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
Anti brdu, by Agilent Technologies (1 mentions)
Lea biotin, by Vector Laboratories (1 mentions)
Sds page, by Beyotime (1 mentions)
12 protocols using anti igf1r
1
Western Blot Analysis of Cell Signaling Proteins
2
Protein Expression Analysis by Western Blot
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Total protein was extracted in RIPA lysis buffer. Proteins extracted from cells were resolved using 10% sodium dodecyl sulfate–polyacrylamide (SDS–PAGE) gel electrophoresis, then transferred to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, USA), blocked in 5% non-fat milk (Sigma–Aldrich, St Louis, MI, USA) for 2 h, and blotted with primary antibody (anti-NEDD4, Cell Signaling Technology, Danvers, MA, USA, 1:1000; anti-IGF-1R, Abcam, 1:1000; anti-PTEN, Cell Signaling Technology, 1:500; anti-p-Aktser473, Cell Signaling Technology, 1:2000; anti-GAPDH, Cell Signaling Technology, 1:2000; anti-β-actin, Sigma–Aldrich, 1:5000) overnight at 4 °C. The next day, membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h at room temperature. Blots were visualized with an ECL detection kit (Millipore) and analyzed using Image J software.
3
Immunohistochemical Analysis of NEDD4 Signaling
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Slides from all patients were stained for NEDD4. Antigen retrieval, blocking procedures, and a modified ImmunoMax method have been previously described [30 (link)]. Briefly, slides were deparaffinized and rehydrated, followed by incubation with 3% hydrogen peroxide and methanol to block endogenous peroxidase activity and non-specific protein–protein interactions. Antigen retrieval was performed with citric acid–based buffer at pH 6.0 using a hot plate in a metal container for 15 min before immunostaining. After 1-h blocking for unwanted staining, primary antibody (anti-NEDD4, 1:500, EMD Millipore, Darmstadt, Germany; anti-IGF-1R, 1:50, Abcam, Cambridge, England; anti-PTEN, 1:100, Cell Signaling Technology, Danvers, MA, USA; anti-p-Aktser473, 1:100, Cell Signaling Technology, Danvers, MA, USA) was added at an optimum dilution. A negative control was prepared by the substitution of primary antibody with phosphate-buffered saline (PBS, 5% BSA). All washing steps were performed with PBS alone, along with PBS with 0.1% Tween. To ensure consistent IHC evaluation, slides from a reference tumor previously defined as positive were included in each staining procedure.
4
BrdU Uptake Assay for Gastric Cell Proliferation
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Gastric epithelial cell proliferation was studied by BrdU uptake. AGS cells were cultured on 6 well-plates (NUNC) by serum free F-12 medium without antibiotics. Cells were incubated with and/or without H. pylori and IL-16 for 24 h, additionally 1 h incubated with 10 µM BrdU (Sigma-Aldrich) solution. After 3 times wash with PBS, cells were lysed with 50 mM NaCl, 25 mM Tris-HCl, 0.5% NP-40, 0.5% sodium deoxycholate and 0.02% sodium azide. Anti-BrdU (10 µm/ml Dako) and anti-IGF-1R (Abcam) antibodies were diluted with 0.01 M phosphate buffer, 0.15 M NaCl pH 7.2, and coated each well of 96-well plates, and incubated at 4°C overnight. 100 µl of samples were pipetted into wells and incubated for 2 h at room temperature after 3 times washing with Buffer B (0.01 M phosphate buffer, 0.50 M NaCl, 0.1% Tween 20, pH 7.2). The plates were blocked with 3% skim milk (Wako) with Buffer B for 30 min after 3 times washing with Buffer B. The plates were incubated with secondary antibodies (Dako) for 1 h, and further incubated with alkaline phosphatase conjugated streptavidin (Dako) for 1 h. The expressions were followed by ampliQ detection kit (K6245, Dako) and determined by a spectrophotometer at 492 nm.
5
Immunoprecipitation and Detection of IGF1R and Integrins
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Antibodies and concentrations used for immunoprecipitations are cataloged in Supplementary Table 2 . Cells were lysed in 2% NP-40 buffer/Buffer A (150 mM NaCl, 0.5 mM Tris, 1 mM EDTA). Lysates were quantified using Bradford reagent. IGF1R, Integrins α4, β1, or β3 were immunoprecipated from at least 200 µg lysate mixed with 40 μl of BSA-blocked protein G-agarose bead slurry (#15920-010, Life Technologies) loaded with 2 µg anti-IGF1R (Abcam), anti-Integrins α4, β1 or β3 antibodies (Cell Signaling). Immunprecipitation was carried out overnight on a rotator at 4 °C, followed by washing with lysis buffer, elution by boiling in Laemmli reducing running buffer and western blot with LEA-biotin (Vector) lectin (for detection of I-branched glycans) or IGF1R, Integrins α4, β1 or β3 antibodies. As controls, immunprecipitations were performed in parallel with equal amounts of respective isotype control antibody.
6
Western Blot Analysis of IGF-1R Expression
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Western blot analysis was performed according to the standard protocol. Following 72-h transfection, transfected cells (miR-592 and NC) were washed and harvested using radioimmunoprecipitation assay lysis buffer (Pierce; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, along with a protease inhibitor (Pierce; Thermo Fisher Scientific, Inc.). Total protein concentration was measured using a bicinchoninic assay protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of protein (20 µg) were then separated by 10% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred to polyvinylidene difluoride membranes (Merck Millipore). Following a blocking incubation with 5% non-fat milk at room temperature for 2 h, the membranes were incubated at 4°C overnight with primary anti-IGF-1R (dilution, 1:1,000; cat no. ab131476; Abcam, Cambridge, MA, USA) and anti-GAPDH (dilution, 1:1,000; cat no. ab201822; Abcam), followed by incubation at room temperature for 1 h with the goat anti-rabbit horseradish peroxidase conjugated secondary antibody (1:3,000 dilution; cat no. ab97051; Abcam). The proteins were detected with an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) and visualized using the FluorChem imaging system (Alpha Innotech, San Leandro, CA, USA). GAPDH was used as an internal control.
7
Immunofluorescence Protocol for IGF1R
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Immunofluorescence was preformed according to the previous methods [20] (link). Cells grown on glass slides (NEST, China) were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and then incubated with the primary antibody anti-IGF1R (1:100, abcam) at 4°C overnight. After being washed in PBS and incubated for 1 hour at 37°C with fluorescence-labeled secondary antibody Alexa Fluor-594 (1:1000, Proteintech, Chicago, USA), the cells were then mounted with prolong gold antifade reagent with DAPI (Invitrogen) for 10 minutes.
8
Osteoblast Differentiation Signaling Pathways
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MC3T3-E1 cells were plated in 6-well plates at a density of 2 × 105 cells/well for 24 h, treated with CAT, ACT, or ECH at a concentration of 10−8 and 10−7 M for 4 h. After removing the medium, cells were lysed in a buffer (P0013 and 1 mM PMSF, Beyotime Institute of Biotechnology, Shanghai, China) for 30 min at 4 °C. The supernatant was harvested by centrifuging at 12,000 g for 5 min at 4 °C. A BCA Kit was used to measure protein concentrations. The proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 5% BSA for 1 h, and then detected for anti-IGF-1R, anti-p-IGF-1R, anti-BMP2, anti-Runx2, anti-Osterix, anti-AKT, anti-p-AKT, anti-PI3K, anti-p-PI3K (Abcam, Cambridge, UK), anti-p-Smad1/5/9, anti-p-mTOR, anti-mTOR, anti-β-actin (Cell Signaling Technology, Beverly, MA, USA), and anti-Smad1 (Boster, Wuhan, China) at 4 °C overnight, and then incubated with anti-goat or anti-rabbit IgG conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. The Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA) was used to scan and analyze the images.
9
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Proteins were collected from cells lysates and loaded to 10% SDS-PAGE gels, and then transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies and then secondary antibodies. The primary antibodies used in this study include anti-Vimentin (Cell Signaling Technology (CST), Rabbit, 1:1000, MA, USA), anti-N-cadherin (CST, Rabbit, 1:1000), anti- IGF1R (CST, Rabbit, 1:1000), anti-GAPDH (Abcam, Mouse, 1:1000), and anti-E-cadherin (Abcam, Rabbit, 1:1000).
10
Protein Expression Profiling of BTICs and hAT-MSCs
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BTICs cells co-cultured with hAT-MSCs were collected and lysed in protein lysis buffer (Cell Signaling, Danvers, MA). Equal amounts of sample lysate were separated by NuPAGE 4–12% bis-Tris gel (Invitrogen) and transferred to a nitrocellulose iblot gel transfer stack (Invitrogen) using the iblot system (Invitrogen). The membranes were blocked with 5% skim milk in Tris-buffered saline, Tween-20 (TBST) buffer and incubated overnight at 4°C with anti-SDF-1 (1:500, Abnova, Taipei, Taiwan), anti-RANTES (1:250, Abcam), anti-IL-6 (1:200, Thermo Scientific, IL), anti-IL-8, anti-IGF-1 (1:500, Abcam), anti-CXCR4R (1:1000, Abcam), anti-CCR5 (1:500, R&D system), anti-IGF1R (1:250, Abcam) and β-actin (1:5000, Sigma-Aldrich). The membranes were washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibodies. The membranes were developed with enhanced chemiluminescence kits (Invitrogen) and exposed to film. For quantification, the band densities were normalized to the levels of β-actin and represented as the relative intensity.
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