Gefitinib (AstraZeneca), Phenylarsine Oxide (PAO) (Sigma‐Aldrich), Filipin III (Sigma‐Aldrich), anti‐EGFR (sc‐373746), anti‐EGFR (4267, Cell signaling), anti‐p‐EGFR (2234, Cell signaling), anti‐STAT3 (sc‐8019), anti‐p‐STAT3 (sc‐8059), anti‐ERK (9102, Cell signaling), anti‐p‐ERK (9101, Cell signaling), anti‐EEA1 (610457, BD Biosciences), anti‐PARP (9542, Cell signaling), anti‐c‐Myc (9402, Cell signaling), anti‐β‐actin (A5316), Goat anti‐mouse IgG (H + L)‐HRP conjugate (1706516, Bio‐Rad), Goat anti‐rabbit IgG polyclonal HRP conjugated (ADI‐SAB‐300, Enzo), Alexa Fluor 488 goat anti‐rabbit antibody (A32731, Thermo Fisher Scientific), and Alexa Fluor 594 goat anti‐mouse antibody (A32742, Thermo Fisher Scientific).
Anti egfr
Manufactured by Cell Signaling Technology
Sourced in United States, United Kingdom, Italy, Germany
Anti-EGFR is a laboratory reagent used for the detection and quantification of the epidermal growth factor receptor (EGFR) protein in various experimental systems. It functions as a specific antibody that binds to EGFR, allowing researchers to analyze the expression and distribution of this important cell signaling protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt, by Cell Signaling Technology (45 mentions)
Anti erk1 2, by Cell Signaling Technology (22 mentions)
Anti erk, by Cell Signaling Technology (20 mentions)
Anti p egfr, by Cell Signaling Technology (18 mentions)
Anti p akt, by Cell Signaling Technology (16 mentions)
Anti phospho egfr, by Cell Signaling Technology (16 mentions)
Anti p erk, by Cell Signaling Technology (15 mentions)
Anti β actin, by Merck Group (15 mentions)
Anti β actin, by Cell Signaling Technology (14 mentions)
Anti gapdh, by Cell Signaling Technology (13 mentions)
Anti cleaved caspase 3, by Cell Signaling Technology (11 mentions)
Anti her2, by Cell Signaling Technology (11 mentions)
Anti phospho erk1 2, by Cell Signaling Technology (11 mentions)
Anti stat3, by Cell Signaling Technology (10 mentions)
Anti phospho akt, by Cell Signaling Technology (10 mentions)
Anti parp, by Cell Signaling Technology (9 mentions)
Anti phospho erk, by Cell Signaling Technology (9 mentions)
Anti β actin, by Santa Cruz Biotechnology (9 mentions)
Anti e cadherin, by Cell Signaling Technology (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
192 protocols using anti egfr
1
Evaluating EGFR Signaling Dynamics
2
Western Blot Analysis of KRAS, EGFR, and GAPDH
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At the end of experimental time-point, the media was aspirated off and the cells were washed twice with ice-cold 1X phosphate-buffered saline (PBS). Cells were then lysed using RIPA buffer (20x volumes of cell pellet; Thermo Fisher Scientific) and protein concentration in extracted whole cell lysate was determined using BCA assay (Thermo Fisher Scientific). Fifty micrograms of lysates were resolved by SDS-PAGE and processed for Western blot using standard methodologies. Blots were probed with the following antibodies: anti-KRAS (clone 9.13, 1:1000; ThermoFisher Scientific), anti-EGFR (#2085; 1:2000; Cell Signaling), anti-EGFR (#2234; 1:1000; Cell Signaling) and anti-GAPDH (#5174, 1:4000; Cell Signaling). Blots were probed with anti-GAPDH antibody to ensure equivalent protein loading across samples.
3
Cell Lysate Preparation and Immunoblotting
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Cell lysates’ preparation and immunoblot analyses were performed as previously reported [35 (link)]. The filters were probed with the indicated primary antibodies: anti-EGFR, anti-PDGFRβ, anti-vinculin and anti-α-tubulin (Cell Signaling Technology Inc.). The blots shown are representative of at least three independent experiments.
4
Cytotoxic Effects of C086 on NSCLC
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The human NSCLC cell lines A549 and NCI-H1975 were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in RPMI-1640 media containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2.
The bacterial strains and plasmids were obtained from the School of Life Science of Xiamen University, China. C086 (purity≥99%) was designed and synthesized by our laboratory (Figure 1B ). C086 was dissolved in DMSO as a stock solution and diluted in culture media. Gefitinib was purchased from LC Laboratories (Woburn, MA, USA). Anti-Hsp90, anti-β-actin, anti-EGFR, anti-Ras, anti-C-Raf, anti-Akt, anti-P-Akt, anti-Mek, anti-P-Mek, anti-Erk½, anti-P-Erk, anti-C-Myc anti-Bax, anti-Bcl-2 (an apoptosis suppression protein), caspase-8, cleaved caspase-8, and the Apoptosis Antibody Sampler Kit containing PARP, cleaved PARP, caspase-9, cleaved caspase-9, caspase-3, cleaved caspase-3, caspase-7, cleaved caspase-7 were purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Annexin-V-APC/PI Apoptosis Detection Kit was purchased from Nanjing Keygen Biotech Co. Ltd (Nanjing, China). Propidium iodide (PI) was obtained from Sigma Aldrich.
The bacterial strains and plasmids were obtained from the School of Life Science of Xiamen University, China. C086 (purity≥99%) was designed and synthesized by our laboratory (
5
Western Blot Analysis of Signaling Proteins
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Cell lysates (40 μg protein/line) were separated on a 10% SDS-PAGE gel and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The blotted membranes were blocked with 5% skim milk or 5% bovine serum albumin and incubated overnight at 4 °C. Anti-PEAK1 (86 kDa, Abnova, Taipei, Taiwan), anti-EGFR (Tyr1173, 175 kDa), anti-EGFR (175 kDa), anti-KRas (21 kDa), anti-phospho-p44/42 Erk (Thr202/Tyr204, 44/42 kDa) anti-p44/42 Erk (44/42 kDa) (Cell Signaling Technology, Danvers, MA, USA), anti-β-actin (43 kDa) and anti-GAPDH (36 kDa) (Abcam, Cambridge, UK) antibodies were used. The detailed procedures are described in the Supplemental materials and methods.
6
Western Blot Analysis of Apoptosis Markers
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All chemicals were sourced from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated. For the western blot analysis, the following primary and secondary antibodies were used: Anti-cleaved caspase-6 (#9761), anti-cleaved caspase-7 (#9491), anti-cleaved caspase-8 (#9496), anti-cleaved caspase-9 (#9505), anti-PARP (#9542), anti-ATF6 (#65880), anti-BiP (#3177), anti-CHOP (#2895), anti-EGFR (#4267), and anti-MEK (#8727) from Cell Signaling Technology (Danvers, MA, USA). Anti-H-FABP (ab45966) and anti-CD36 (ab133625) from Abcam (Cambridge, UK), and anti-actin (clone AC-40).
7
HNSCC Cell Culture and Reagents
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Cal27 and SCC25 cells were obtained from American Type Culture Collection. OSC19 cells were kindly provided by Dr. Jeffrey Myers (The University of Texas MD Anderson Cancer Center, Houston, TX, USA). Cells were grown in Dulbecco’s Modified Eagle’s Medium/F12 supplemented with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C and 5% CO2. As extended in vitro cell culture and clonal expansion can lead to the emergence of new genotypes and altered cellular phenotypes over time, all experiments were performed on early passage cells between passage number 3 and 10. Chemical reagents were obtained as follows: cisplatin (purity >99.9%), crystal violet, and sodium orthovanadate from Sigma-Aldrich (St. Louis, MO, USA); gefitinib (purity >99%) from AstraZeneca (Macclesfield, UK); recombinant human epidermal growth factor (rhEGF) from Cell Sciences (Canton, MA, USA); alamarBlue and trypan blue from Invitrogen (Carlsbad, CA, USA); anti-EGFR, anti-phosphorylated EGFR (pEGFR), anti-ERK1/2, anti-pERK1/2, anti-poly ADP ribose polymerase (PARP), anti-β-actin, and anti-γH2AX from Cell Signaling (Beverly, MA, USA); annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) from Millipore (Bedford, MA, USA); and dimethylsulfoxide (DMSO), dithiothreitol (DTT), FBS and methanol from Thermo Fisher Scientific (Waltham, MA, USA).
8
Western Blot Analysis of Protein Signaling
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Samples were denatured in buffer containing 60 mM Tris/pH 6.8, 25% glycerol, 2% sodium dodecyl sulfate (SDS), and 14.4 mM 2-mercaptoethanol with 0.1% bromophenol blue, and boiled for 5 minutes. SDS polyacrylamide gels (Bio-Rad, Hercules, CA, USA) were loaded with 25 µg of total protein per lane. Prestained molecular weight markers (Bio-Rad) were used as standards. Electrophoresed samples were transferred to polyvinylidene difluoride membranes (GE Healthcare, Piscataway, NJ, USA). After transfer, membranes were blocked with 3% bovine serum albumin in Tris-phosphate-buffered saline (TPBS; 200 mM Tris/pH 7.0, 1.37 M NaCl, 1% Tween-20) for 1 hour at room temperature. Membranes were then incubated with anti-RET, anti-phospho-RET, anti-EGFR, anti-phospho-EGFR, anti-AKT, anti-phospho-AKT, anti-ERK, anti-phospho-ERK, or anti-beta actin (1:1000; Cell Signaling Technology, Danvers, MA, USA) overnight at 4℃, washed as before, and incubated with goat anti-rabbit peroxidase-conjugated secondary antibody (1:4000; Cell Signaling Technology) for 2 hours at room temperature. Membranes were then washed, and expressed proteins were detected with Pierce ECL plus western blotting substrate (Pierce, Rockford, IL, USA). Experiments were conducted at least three times independently.
9
Protein Analysis of MM1.S Cells
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Total proteins from MM1.S cells lysates, MM1.S exosome, conditioned medium of cells deprived of exosomes, patient’s exosomes, and RAW 264.7 lysates were extracted and analyzed by SDS-PAGE followed by western blotting. Antibodies used in the experiments were as follows: anti-EGFR, pEGFR (Cell Signalling Technology, Lane Danvers, MA, USA), anti-AREG (Novus Biologicals), and anti-GAPDH (Santa Cruz Biotechnology, CA, USA).
10
Duodenal Biopsy and Organoid Protein Analysis
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Biopsy fragments from duodenum obtained from GCD–CeD and organoids, were processed [10 (link),28 (link)] After removing matrigel by Cell Recovery Solution (cat. 354270, Corning Milan Italy), organoids were homogenized in 50 µL tissue homogenization buffer [28 (link)]. The cell lysates (10 μg/mL) were analysedby SDS-PAGE and transferred onto nitrocellulose membranes by Trans Blot Turbo (cat.1704158, BioRad, Milan, Italy). The membranes were blocked with 5% non-fat dry milk and probed with anti-PTPRk (cat. ab222249, Abcam, Microtech, Naples, Italy) and rabbit anti-pY-ERK1/2, (cat.20869, Elabscience, Microtech, Naples, Italy), anti-P-EGFR (Y1068) (cat.3777,Cell Signalling, Euroclone Milan, Italy) and anti-EGFR (cat.2232, Cell Signalling, Euroclone Milan, Italy), mouse anti-GAPDH, (cat. G8795, Sigma-Aldrich, Milan, Italy) and with rabbit anti-ERK1/2 (cat.31374, Elabscience, Microtech, Naples, Italy) were used as loading controls for biopsies and organoids, respectively. Then, two 10 min exposure using ECL (cat. RPN2209, GE Healthcare, Amersham, Buckinghamshire, UK) allowed to visualise the bands of interest. The bands intensity was evaluated by integrating all the pixels of the band after subtraction of the background to calculate the average of the pixels surrounding the band.
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