Protein extraction and Western blotting were performed as previously described (24 (link)). Primary antibodies against IL17RD, FLAG, pAKT, pERK, pERBB2 EGFR, pEGFR, and beta actin were used with a dilution of 1:500 for IL17RD (R and D systems), 1:1000 for FLAG (Clontech, Mountain View, CA),1:500 pAKT (Cell Signaling Technology, Danvers, MA), 1:500 for EGFR (Cell Signaling Technology), 1:100 for pEGFR (Santa Cruz Biotechnology, Dallas, TX), 1:2500 for pERK (Santa Cruz Biotechnology), 1:200 for pERBB2 (Santa Cruz Biotechnology) and 1:5000 for beta actin (Sigma Aldrich, St. Louis, MO). Mean expression between treatment group was compared using a Student’s t-test.
P egfr
Manufactured by R&D Systems
Sourced in United States
P-EGFR is a recombinant human phosphorylated epidermal growth factor receptor (EGFR) protein. It is a cytoplasmic domain of the EGFR protein that contains tyrosine phosphorylation sites.
Automatically generated - may contain errors
Lab products found in correlation
P akt, by Cell Signaling Technology (2 mentions)
Il 17rd, by R&D Systems (1 mentions)
P akt, by R&D Systems (1 mentions)
β actin, by Merck Group (1 mentions)
Flag tag (flag), by R&D Systems (1 mentions)
P erk, by R&D Systems (1 mentions)
P erbb2, by Santa Cruz Biotechnology (1 mentions)
P egfr, by Santa Cruz Biotechnology (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Sybr green pcr master mix, by Roche (1 mentions)
Applied biosystems 7500, by Thermo Fisher Scientific (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript reverse transcription reagent kit with gdna eraser, by Takara Bio (1 mentions)
P mapk, by Cell Signaling Technology (1 mentions)
Adam9, by R&D Systems (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
P ikkα β, by Cell Signaling Technology (1 mentions)
Ecl plu, by Cytiva (1 mentions)
3 protocols using p egfr
1
Protein Expression Analysis via Western Blotting
2
Comprehensive Western Blot and RT-qPCR Analysis
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Western blotting was performed as described previously (15 (link)). All the types of cells were lysed by RIPA buffer with cocktail protease and phosphatase inhibitors (Thermo Fisher Scientific). Total protein extracts (40 μg) were subjected to western blotting analysis with antibodies against the following proteins: ADAM9 (R&D system); p-EGFR (Tyr1068), EGFR, p-MAPK (Tyr202/204), MAPK, GAPDH, p-AKT (Ser473), AKT, p-IKKα/β (Ser176/180), IKKβ, p-NF-κBp65 (Ser536), NF-κBp65, p-IκBα (Ser32), IκBα, and β-actin (Cell Signaling, Massachusetts, USA).
Total RNA was extracted from all the types of cells by using TRIzol Reagent (Thermo Fisher Scientific) according to the vendor's instruction. The cDNA synthesis was achieved by using PrimeScript reverse transcription reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). Quantitative PCR was performed with ADAM9 and GAPDH primers by using SYBR Green PCR Master Mix (Roche, Baden-Württemberg, Germany) in a real-time PCR System (Applied Biosystems 7500, Thermo Fisher Scientific). Primer sequences were as follows: ADAM9, 5′-CCTCGGGGACCCTTCGTGT-3′ and 5′-ATCCCATAACTCGCATTCTCTAAA-3′; GAPDH, 5′-CCACCCATGGCAAATTCCATGGCA-3′ and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. Quantitative analysis of RT-qPCR was achieved by the 2−ΔΔCt method.
Total RNA was extracted from all the types of cells by using TRIzol Reagent (Thermo Fisher Scientific) according to the vendor's instruction. The cDNA synthesis was achieved by using PrimeScript reverse transcription reagent Kit with gDNA Eraser (TaKaRa, Shiga, Japan). Quantitative PCR was performed with ADAM9 and GAPDH primers by using SYBR Green PCR Master Mix (Roche, Baden-Württemberg, Germany) in a real-time PCR System (Applied Biosystems 7500, Thermo Fisher Scientific). Primer sequences were as follows: ADAM9, 5′-CCTCGGGGACCCTTCGTGT-3′ and 5′-ATCCCATAACTCGCATTCTCTAAA-3′; GAPDH, 5′-CCACCCATGGCAAATTCCATGGCA-3′ and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. Quantitative analysis of RT-qPCR was achieved by the 2−ΔΔCt method.
3
Western Blotting Analysis of EGFR Expression
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Western blotting analysis was conducted in order to analyze the degree of protein expression by using a MDA-MB-468 cytolysate. First, we boiled the protein in a Laemmli sample buffer for about 5 minutes and then it was electrophoresed in 8% sodium dodecyl sulfate polyacrylamide gel to pass the protein to the polyvinylidene fluoride membrane, and then the movement of the nonspecific protein was blocked by using 10% skim milk for 15 minutes in Tris buffered saline (TBS) that also included 0.01% Tween-20. All the blots were cultured for about 12 hours at 4℃ with anti-t-epidermal growth factor receptor (EGFR), p-EGFR (R&D System, Minneapolis, MN, USA) primary antibody and then washed, three times, in TBS/T and reacted again in TBS/T buffer solution with antirabbit peroxidase-conjugated antibody (1/2,000 dilution), a secondary antibody. After being cultured for about 1 hour at room temperature, the blots were washed in TBS/T buffer solution three times and ECLplus (Amersham, Buckinghamshire, UK) reagent was used for development.
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