Brains were fixed by transcardiac perfusion with 4% paraformaldehyde (PFA)/PBS, postfixed overnight at 4 °C, cryoprotected in 30% sucrose/PBS, and cryosectioned. For IGF-1 immunofluorescence, sections were processed to citric acid antigen retrieval prior to immunolabeling. Sections were blocked in 1% BSA and 0.2% Triton X-100 in PBS and incubated with primary antibodies overnight at 4 °C. Sections were incubated with appropriate fluorescent secondary antibodies (1:250; Invitrogen) 1 h at room temperature and mounted with Vectashield antifade mounting agent (Vector). All antibodies were diluted in 1% BSA and 0.2% Triton X-100/PBS. Primary antibodies used were anti-TH (1:1,000; Pel-Freez P40101-150 and Millipore AB1542), anti-IGF-1 (1:200, AF-291-NA; R&D Systems), anti–IGF-1R (1: 200, 9750; Cell Signaling), anti-phosphoSer40 TH (1:1,000, AB5935; Merck), anti-phosphoTyr1135/1136 IGF-1R (1:200, 3024; Cell Signaling), and anti-phosphoSer235/236 S6 Ribosomal protein (1:100, 4858; Cell Signaling).
Anti igf 1r
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-IGF-1R is a laboratory reagent used to detect and quantify the expression of the insulin-like growth factor 1 receptor (IGF-1R) in biological samples. It is a highly specific antibody that binds to IGF-1R and can be used in various analytical techniques, such as Western blotting, immunohistochemistry, and ELISA.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (14 mentions)
Anti phospho akt, by Cell Signaling Technology (8 mentions)
Anti erk1 2, by Cell Signaling Technology (6 mentions)
Anti egfr, by Cell Signaling Technology (5 mentions)
Anti phospho igf 1r, by Cell Signaling Technology (5 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Anti p akt, by Cell Signaling Technology (3 mentions)
Cisplatin, by Merck Group (3 mentions)
Anti p igf 1r, by Cell Signaling Technology (3 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (3 mentions)
Anti ir, by Cell Signaling Technology (3 mentions)
Anti β actin, by Santa Cruz Biotechnology (3 mentions)
Exoab cd63a 1, by System Biosciences (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Anti β actin antibody, by Cell Signaling Technology (2 mentions)
Anti cleaved caspase 8, by Cell Signaling Technology (2 mentions)
Anti bax, by Cell Signaling Technology (2 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
40 protocols using anti igf 1r
1
Immunofluorescence Staining of Brain Tissue
2
Protein Expression Analysis in Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were homogenized in hypotonic buffer with Triton X-100 while using metal beads in a Tissue Lyser (Qiagen, Hilden, Germany) as previously described [19 (link)]. 40–50 μg of total protein was resolved per lane by SDS-PAGE and transferred to a nitrocellulose membrane. Blots were probed with specific antibodies against GRK2 (sc-562, Santa Cruz Biotechnology, Dallas, TX, USA), β-Arrestin 1 and 2 [20 (link)], anti-pAkt (Ser473 #9271, Cell Signalling, Danvers, MA, USA), total Akt (#9272 Cell Signalling), β-Actin (127M4866V, Sigma, San Luis, MO, USA), GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase, sc-32233, Santa Cruz), anti-phospho-tyrosine (#61-5800, Invitrogen, Carlsbad, CA, USA), anti-insulin receptor β subunit (sc-57342, Santa Cruz), anti IGF1R (#9750, Cell Signalling), and α-Tubulin (sc-53030, Santa Cruz). Immunoreactive bands were visualized while using enhanced chemiluminescence (ECL; Amersham Biosciences, Buckinghamshire, UK) or the Odyssey Infrared Imaging System (Li-Cor Biosciences, Lincoln, NE, USA). Films were scanned with a GS-700 Imaging Densitometer and then analyzed with Quantity One Software (Bio-Rad, Hercules, CA, USA), or using an Odyssey Classic reader and the Odyssey software package 3.0 (Li-Cor Biosciences).
3
Immunoblotting Antibody Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies raised against EGFR (sc-03), Gi1α (sc-391), Gi2α (sc-7276), Gi3α (sc-262), AKT1/2 (sc-8312), ERK1/2 (sc-94), FRS2 (sc-8318) and SH-PTP2 (Shp2, sc-280) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-β-actin antibody was purchased from Sigma-Aldrich (St. Louis, Mo). Anti-EGFR, anti-FGFR, anti-Gab1, anti-IGF-1R, and other anti-phospho antibodies were purchased from Cell Signaling Technology (Danvers, MA).
4
Western Blotting Protocol for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as described previously.13 (link) The anti-IGF-1R, anti-IR, and Akt and p-Akt antibodies (Cell Signaling) were used at 1:1,000 dilutions. The p-Erk1/2 and Erk1/2 antibodies (both from Cell Signaling) were used at 1:2,000 dilutions. The anti-GLP-1R (Sigma, St Louis, MO, USA) was used at 8 μg/mL. The control anti-β-actin antibody was diluted at 1:1,000 (Zhongshan Golden Bridge, Beijing, China).
5
Comprehensive Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed using RIPA buffer (50 mM Tris HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 0.25% deoxycholate) supplemented with protease inhibitor tablets (Roche Molecular Biochemicals) and phosphatase inhibitors (1 mM NaF, 1 mM Na3VO4 and 1 mM β-glycerophosphate). Cell lysates were cleared by centrifugation (13,000 × g, 15 min, 4 °C), and protein concentration was determined by the Bradford method (Bio-Rad protein Assay). Proteins samples were separated on SDS-PAGE, and membranes were incubated with primary antibodies overnight at 4 °C on a shaker with the following primary antibodies: anti-IGF-1R (Cell Signaling), anti-IR-α (Cell Signaling), anti-EGFR (Cell Signaling), anti-furin (Proteintech), anti-Akt and anti-pAkt (Cell Signaling), anti-ERK and anti-pERK (Cell Signaling), anti-S6 and anti-pS6 (Cell Signaling), anti-S6K1 and anti-pS6K1 (Cell Signaling), anti-AMPK and anti-pAMPK (Cell Signaling) and anti-Tubulin (Sigma). Membranes were washed with TBS-Tween, and incubated with anti-rabbit or anti-mouse secondary antibodies. Detection was carried out using ECL Prime reagent (GE Healthcare).
6
Quantification of Pancreatic Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pancreatic tissues were processed as described [32 (link)]. In brief, mouse pancreases were dissected and fixed in 4% formaldehyde at 4 °C for 12 h before embedding in paraffin. Sections of 5 μm were deparaffinized, rehydrated, and incubated with anti-insulin (Thermo Scientific), GRP94 (Enzo), or anti-IGF-1R (Cell signaling) antibodies overnight at 4 °C, followed by fluorescein isothiocyanate (FITC), Cy3- or Cy5-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). Slides were mounted with VECTASHIELD mount media with 4′6-diamidino-2-phenylindole (DAPI) (Vector Labs). Fluorescence was analyzed using a Zeiss Axio Imager M2 microscope (Carl Zeiss, Inc.), and images were quantified using ImageJ software. Corrected total cell fluorescence (CTCF) of GRP94 fluorescence was quantified using the ImageJ software. The CTCF = Integrated Density – (Area of selected cell X Mean fluorescence of background readings). Quantitative analyses of the pancreatic area were performed on an Olympus BX40 microscope using the Olympus microscopy Image system.
7
Immunoblotting Analysis of EGFR, ERK, and AKT
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A549 and PC-9 cells were lysed in buffer containing proteinase inhibitors. The total protein of cells was obtained using the Nuclear Extract Kit (Active Motif Corp, USA). Protein concentration was determined by the BCA protein assay (Pierce, Rockford, IL, USA). Samples containing 100 µg of total protein were electrophoresed on 10% SDS-PAGE and transferred onto a nitrocellulose membrane by electroblotting. The blots were probed using the following antibodies: anti-EGFR (1∶1000; Santa Cruz, CA), anti-phosphorylated-EGFR (1∶1000), anti-ERK1/2 (1∶600), anti-phosphorylated-ERK1/2 (1∶600), anti-AKT (1∶1000), anti-phosphorylated-AKT (1∶1000), anti-IGF-1R (1∶600), anti-phosphorilated-IGF-1R (1∶600) and anti-β-actin antibody (1∶800) (all the above antibodies were from Cell Signaling Technology, USA), followed by incubation with horseradish peroxidase (HRP) conjugated goat-anti-mouse secondary antibody (Santa Cruz, CA, USA). The blots were visualized by an enhanced chemiluminescence kit (ECL) (Amersham Pharmacia Biotech, Arlington Heights, IL, USA) according to the manufacturer’s instructions. Each experiment was performed in triplicate.
8
Pharmacological Inhibition of Enzalutamide and MK2206 on Prostate Cancer
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IU1 (S7134), enzalutamide (S1250) and MK2206 (S1078) were obtained from Selleckchem (Houston, TX, USA). DMSO was used to dissolve these inhibitors and the inhibitors were stored at − 20 °C. USP14 (sc-76,817) siRNA was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MTS (catalog no. G111) was obtained through Promega Corporation (Madison, WI, USA). Annexin V-FITC/PI apoptosis detection kits of Keygen Company (Nanjing, China)(KGA107) were purchased. Cell Lysis Buffer (#9803) was from Cell Signaling Technology (MA, USA) and stored at − 20 °C. Anti-GAPDH (MB001) was obtained from Bioworld Technology (St.Louis Park, MN, USA). The other antibodies were from Cell Signaling Technology (MA, USA): anti-PARP (#9542), anti-caspase 3 (#9668), anti-cleaved caspase 3 (#9661), anti-caspase 8 (#9746), anti-cleaved caspase 8 (#9496), anti-cleaved caspase 9 (#9501), anti-Bax (#5023), anti-CDK4(#12790), anti-P27(#3686), anti-caspase 9 (#9508), anti-CDK2 (#2546), anti-CyclinD1(#2922), anti-total-AKT (#9272), anti-AR (#5153), anti-USP14 (#11931), anti-Bcl-2 (#15071), anti-GSK3β (#12456), anti-p-GSK3β (Ser9)(#9323), anti-phospho-AKT (Ser473)(#4060), anti-β-catenin (#8480), anti-EGFR (#4267), anti-IGF1R (#9750).
9
Western Blotting Quantification Techniques
10
Quantification of Bioactive IGF-1 and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblotting was performed as described [30 (link), 31 (link)]. The levels of free bioactive IGF-1 were determined using IGF-I Bioactive ELISA (AnshLabs). The following antibodies were used: rabbit polyclonal anti-phospho-Akt (9271, Cell Signaling Technologies); rabbit polyclonal anti-CD31 (ab28364, Abcam); mouse monoclonal anti- FLAG (M2, Sigma-Aldrich); rabbit polyclonal anti- FLI-1 (ab15289, Abcam); mouse monoclonal anti-HIF- 1α (NB100-131, Novus); rabbit polyclonal anti-IGFBP2 (3922, Cell Signaling Technologies); goat polyclonal anti-IGFBP4 (AF804, R&D Systems); mouse monoclonal anti-IGFBP5 (sc-515116, Santa Cruz Biotechnology); rabbit monoclonal anti-IGF-1R (9750, Cell Signaling Technologies); rabbit monoclonal anti-phospho-IGF-1R (3918, Cell Signaling Technologies); goat polyclonal anti- PGK1 (sc-17943, Santa Cruz Biotechnologies); and mouse monoclonal anti-tubulin (DM1A, Thermo Scientific);
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!