Antibodies used included: EGFR (1:2000, #4267), phospho-EGFR (Y992) (1:1000, #2235), phospho-EGFR (Y1068) (1:1000, #2234), phospho-EGFR (Y1173) (1:1000, #4407) (For EGFR phosphorylation sites, we utilized codon numbering of mature EGFR sequence that does not include the 24-residue signal sequence, Supplementary Table 1 ), horseradish peroxidase (HRP)-conjugated anti-mouse (1:5000, #7076), and HRP-conjugated anti-rabbit (1:5000, #7074) (Cell Signaling, Beverly, MA); V5 (1:5000, MCA1360GA, AbD Serotec), Myc (1:2500, Sigma-Aldrich A5963); actin antibody (1:5000, Sigma-Aldrich A2066).
Phospho egfr
Manufactured by Cell Signaling Technology
Sourced in United States
Phospho-EGFR is a lab equipment product that detects the phosphorylated form of the Epidermal Growth Factor Receptor (EGFR) protein. It is used to measure the activation state of EGFR in cellular samples.
Automatically generated - may contain errors
Lab products found in correlation
Phospho akt, by Cell Signaling Technology (41 mentions)
Phospho erk1 2, by Cell Signaling Technology (17 mentions)
Phospho erk, by Cell Signaling Technology (15 mentions)
β actin, by Cell Signaling Technology (13 mentions)
Erk1 2, by Cell Signaling Technology (10 mentions)
Caspase 3, by Cell Signaling Technology (9 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Total egfr, by Cell Signaling Technology (9 mentions)
Cleaved caspase 3, by Cell Signaling Technology (8 mentions)
Stat3, by Cell Signaling Technology (6 mentions)
Phospho mek1 2, by Cell Signaling Technology (6 mentions)
Phospho stat3, by Cell Signaling Technology (5 mentions)
Vimentin, by Cell Signaling Technology (5 mentions)
Phospho erbb2, by Cell Signaling Technology (5 mentions)
Cleaved parp, by Cell Signaling Technology (5 mentions)
Ripa buffer, by Thermo Fisher Scientific (5 mentions)
Total akt, by Cell Signaling Technology (4 mentions)
Phospho p38, by Cell Signaling Technology (4 mentions)
Phospho met, by Cell Signaling Technology (4 mentions)
E cadherin, by Cell Signaling Technology (4 mentions)
85 protocols using phospho egfr
1
Quantifying EGFR Phosphorylation Sites
2
Western Blot Analysis of EGFR Signaling
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Cells were lysed in RIPA buffer (Tris pH 7.4 50 mM, NaCl 150 mM, NP-40 1%, SDS 0.1%, EDTA 2 μM) containing proteinase inhibitors (Roche) and phosphatase inhibitors (Roche). The cell lysates (20 μg protein) were subjected to SDS-PAGE and Western blot. Antibodies against the following proteins were used: phospho-EGFR (Y1068), phospho-EGFR (Y845), EGFR, phospho-MEK1/2 (S217/221), MEK1/2, phospho-ERK (T202/Y204), ERK, phospho-AKT (S473), AKT, ERBB3, INSR, DUSP4, DUSP6, Rab11, E-Cadherin, PTEN, AXL, HER2, CRKL, MET, IGF-1R, ARAF, BRAF, phospho-CRAF (S338), CRAF, NRAS, and Actin (Cell Signaling Technology).
3
Western Blot Analysis of Signaling Proteins
4
Western Blot Analysis of Protein Signaling
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Protein lysates were prepared in RIPA lysis buffer, and separated on 10% SDS–polyacrylamide gel electrophoresis gels by electrophoresis. Subsequently, the lysates were transferred to nitrocellulose membranes and probed with the following antibodies and concentrations: HIF-2α 1:1,000 (Novus #NB100-122), HIF-1α 1:1,000 (Cayman Chemicals #1006421), caspase-3 1:1,000 (Cell Signaling #9662 Danvers, MA, USA), GAPDH 1:2,000 (Cell Signaling #2118), β-tubulin 1:1,500 (Cell Signaling #2146), phospho-4E-BP1 1:1,000 (S65, Cell Signaling #9451), 4E-BP1 1:1,000 (Cell Signaling #9452), phospho-S6K1 1:1,000 (T389, Cell Signaling #9205), c-Myc 1:5,000 (Abcam #32072), S6K1 1:1,000 (Cell Signaling #2708), phospho-AKT 1:1,000 (S473, Cell Signaling #9271), AKT 1:1,000 (Cell Signaling #9272), phospho-EGFR 1:1,000 (Y1068, Cell Signaling #3777), EGFR 1:1,000 (Cell Signaling #4267) ANO1 1:500 (Abcam ab64085), phospho-CAMKIIα 1:1,000 (T286, Cell Signaling #12716) and CAMKII 1:1,000 (Cell Signaling #11945). Uncropped immunoblot images are included in Supplementary Fig. 8 .
5
Investigating GL-1196 Protein Expression
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To investigate the effect of GL-1196 on protein expression, whole cell extracts were prepared from 1 × 106 cells in RIPA lyses buffer (150 mM NaCl, 50 mM Tris/HCl, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 0.25% Na-deoxycholate and protease inhibitor cocktail). Equal amounts of denatured protein were next separated on SDS-PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membrane was blocked in 5% skim milk in TBS-T (137 mM NaCl, 20 mM Tris, pH 7.4, 0.05% Tween-20) for 3 h at room temperature, and the proteins were subsequently incubated with specific antibodies: PAK4, phospho-PAK4 Ser474/PAK5 Ser602/PAK6 Ser560, LIMK1, phospho-LIMK1 Thr508/LIMK2 Thr505, phospho-EGFR Tyr845, cofilin, phospho-cofilin Ser3,c-Src, phosphor-c-Src Tyr416, CDK4, CDK6 (Cell signal, Danvers, MA, USA), cyclinD1 (Neomarker, Waltham, MA, USA), EGFR (Bioworld, Louis Park, MN, USA). All PVDF membranes were detected by chemiluminescence (ECL, Pierce Technology, Waltham, MA, USA). To guaranteethe equal loading amount, membranes were stripped and reprobed with GAPDH antibody (Shang Hai Kangchen, Shanghai, China).
6
Rabbit Antibody Immunoblotting Protocol
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Rabbit antibodies specific for EGFR, phospho-EGFR, and β-actin were obtained from Cell Signaling (Beverly, MA).
7
Characterization of NSCLC Cell Lines
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Human NSCLC cell lines NCI-H1975 (Cat#: CRL-5908), NCI-H1650 (Cat#: CRL-5883), and A549 (Cat#: CRM-CCL-185) were purchased from the American Type Culture Collection. Authenticity of NCI-H1975 was certified by STR sequencing analysis (Biowing Biotechnology Co. Ltd., Shanghai). A549 cells were cultured in DMEM (Gibco, ThermoFisher Scientific, Cat#: 12100061), while other cells were maintained in RPMI 1640 (Gibco, ThermoFisher Scientific, Cat#: 31800089). All culture media were supplemented with 10% fetal bovine serum (Gibco, ThermoFisher Scientific, Cat#: 10270098). Cells were maintained in a humidified atmosphere with 5% CO2 at 37°C in incubators.
Erlotinib was purchased from Cayman Chemical (Cat#: 10483). Gefitinib was purchased from SelleckChem (Cat#: S1025). Anti-ADAM17 primary antibody was ordered from Abcam (Cat#: ab39162). The primary antibodies against α-tubulin (Cat#: A11126) or GAPDH (Cat#: MA5-15738) were purchased from Invitrogen, while all other antibodies, including phospho-EGFR (Cat#: 2236S), EGFR (Cat#: 4267S), phospho-ERK (Cat#: 9101S), ERK (Cat#: 9102S), and β-actin (Cat#: 4967), were purchased from Cell Signaling Technology. Control human plasma IgG was purchased from R&D Systems (Cat#: 1-001-A). All other chemicals were purchased from Sigma or Sigma-Aldrich.
Erlotinib was purchased from Cayman Chemical (Cat#: 10483). Gefitinib was purchased from SelleckChem (Cat#: S1025). Anti-ADAM17 primary antibody was ordered from Abcam (Cat#: ab39162). The primary antibodies against α-tubulin (Cat#: A11126) or GAPDH (Cat#: MA5-15738) were purchased from Invitrogen, while all other antibodies, including phospho-EGFR (Cat#: 2236S), EGFR (Cat#: 4267S), phospho-ERK (Cat#: 9101S), ERK (Cat#: 9102S), and β-actin (Cat#: 4967), were purchased from Cell Signaling Technology. Control human plasma IgG was purchased from R&D Systems (Cat#: 1-001-A). All other chemicals were purchased from Sigma or Sigma-Aldrich.
8
EGFR Mutant Cell Signaling Profiling
9
EGF Stimulation of A431 Cells
10
Xenograft Tumor Model in BALB/c Nude Mice
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The A549 cells carrying the pcDNA3.1/luciferase vector (1 × 106/100 μl PBS) were inoculated subcutaneously (s.c.) into 6-week-old female BALB/c nude mice (BALB/cAnN.Cg-Foxn1nu/CrlNarl; n=11), and tumors were allowed to develop for 8 weeks. A randomized method was used to assign the mice into the experimental groups (control and MCT-1). The animal studies were conducted in accordance with the Animal Use Protocol approved by the National Health Research Institutes (NHRI-IACUC-104020-A). Tumor tissues were processed for immunohistochemistry analysis as previously described.27 (link) Luciferin (150 mg/kg; PerkinElmer, Waltham, MA, USA) was intraperitoneally injected into mice to detect tumor progression using a Xenogen IVIS 200 bioluminescence imaging system (Caliper LifeSciences, Hopkinton, MA, USA).
The Abs for immunohistochemistry were diluted as follows: MCT-1, 1:500 (GeneTex, GTX117793); YY1, 1:400 (GeneTex, GTX62783); CD31, 1:200 (Abcam, ab28364); CD163, 1:100 (Abcam, ab189915); αSMA, 1:4000 (GeneTex, GTX112862); MnSOD, 1:2000 (Enzo Life Sciences, Inc., ADI-SOD-111-D); p53, 1:100 (Millipore, DAM1698716, Billerica, MA, USA); and phospho-EGFR, 1:100 (Cell Signaling Technology, #4407S).
The Abs for immunohistochemistry were diluted as follows: MCT-1, 1:500 (GeneTex, GTX117793); YY1, 1:400 (GeneTex, GTX62783); CD31, 1:200 (Abcam, ab28364); CD163, 1:100 (Abcam, ab189915); αSMA, 1:4000 (GeneTex, GTX112862); MnSOD, 1:2000 (Enzo Life Sciences, Inc., ADI-SOD-111-D); p53, 1:100 (Millipore, DAM1698716, Billerica, MA, USA); and phospho-EGFR, 1:100 (Cell Signaling Technology, #4407S).
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