The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
1/1000 - 1/10000. Detects a band of approximately 170 kDa (predicted molecular weight: 134 kDa).
Use at an assay dependent concentration.
1/100 - 1/250.
Is unsuitable for Flow Cyt or IHC.
Receptor tyrosine kinase binding ligands of the EGF family and activating several signaling cascades to convert extracellular cues into appropriate cellular responses. Known ligands include EGF, TGFA/TGF-alpha, amphiregulin, epigen/EPGN, BTC/betacellulin, epiregulin/EREG and HBEGF/heparin-binding EGF. Ligand binding triggers receptor homo- and/or heterodimerization and autophosphorylation on key cytoplasmic residues. The phosphorylated receptor recruits adapter proteins like GRB2 which in turn activates complex downstream signaling cascades. Activates at least 4 major downstream signaling cascades including the RAS-RAF-MEK-ERK, PI3 kinase-AKT, PLCgamma-PKC and STATs modules. May also activate the NF-kappa-B signaling cascade. Also directly phosphorylates other proteins like RGS16, activating its GTPase activity and probably coupling the EGF receptor signaling to the G protein-coupled receptor signaling. Also phosphorylates MUC1 and increases its interaction with SRC and CTNNB1/beta-catenin. Isoform 2 may act as an antagonist of EGF action.
Ubiquitously expressed. Isoform 2 is also expressed in ovarian cancers.
Involvement in disease
Lung cancer Inflammatory skin and bowel disease, neonatal, 2
Belongs to the protein kinase superfamily. Tyr protein kinase family. EGF receptor subfamily. Contains 1 protein kinase domain.
Phosphorylation at Ser-695 is partial and occurs only if Thr-693 is phosphorylated. Phosphorylation at Thr-678 and Thr-693 by PRKD1 inhibits EGF-induced MAPK8/JNK1 activation. Dephosphorylation by PTPRJ prevents endocytosis and stabilizes the receptor at the plasma membrane. Autophosphorylation at Tyr-1197 is stimulated by methylation at Arg-1199 and enhances interaction with PTPN6. Autophosphorylation at Tyr-1092 and/or Tyr-1110 recruits STAT3. Dephosphorylated by PTPN1 and PTPN2. Monoubiquitinated and polyubiquitinated upon EGF stimulation; which does not affect tyrosine kinase activity or signaling capacity but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63', but linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occurs. Deubiquitination by OTUD7B prevents degradation. Ubiquitinated by RNF115 and RNF126. Methylated. Methylation at Arg-1199 by PRMT5 stimulates phosphorylation at Tyr-1197.
Secreted and Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus membrane. Nucleus membrane. Endosome. Endosome membrane. Nucleus. In response to EGF, translocated from the cell membrane to the nucleus via Golgi and ER. Endocytosed upon activation by ligand. Colocalized with GPER1 in the nucleus of estrogen agonist-induced cancer-associated fibroblasts (CAF).
Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma cell line) cells labeling EGFR (phospho Y1086) with ab32086 at 1/100, 3 μg/ml. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077, a AlexaFluor®488 Goat anti-Rabbit IgG was used as the secondary antibody at 1/1000, 2 μg/ml. Cells were counterstained with ab195889, anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200, 2.5 μg/ml. Nuclear stain was DAPI (blue).
The green staining on the membrane was increased in the EGF (100ng/ml, 10min) treated A431 cells when compared with A431 cells without treatment. After LP treatment, the green signaling was obviously decreased.
For the pan antibody, there was no great difference after EGF (100ng/ml, 10min) or EGF (100ng/ml, 10min) + LP treatment. The data showed mostly membranous staining.
Dot blot - Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086)
Dot blot analysis of EGFR (phospho Y1086) phospho peptide (Lane 1), EGFR non-phospho peptide (Lane 2), labeling EGFR (phospho Y1086) with ab32068 at a dilution of 1/1000. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) was used as the secondary antibody at a dilution of 1/100000.
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 5 seconds.
Western blot - Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086)
All lanes : Anti-EGFR (phospho Y1086) antibody [Y39] (ab32086) at 1/3000 dilution
Lane 1 : Untreated A431 (Human epidermoid carcinoma cell line) whole cell lysates Lane 2 : A431 (Human epidermoid carcinoma cell line) treated with 100 ng/ml EGF for 10 minutes whole cell lysates
Lysates/proteins at 15 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution
Hashikawa KI et al. Dysfunction of the circadian transcriptional factor CLOCK in mice resists chemical carcinogen-induced tumorigenesis. Sci Rep7:9995 (2017).
Read more (PubMed: 28855649) »
Lantermann AB et al. Inhibition of Casein Kinase 1 Alpha Prevents Acquired Drug Resistance to Erlotinib in EGFR-Mutant Non-Small Cell Lung Cancer. Cancer Res75:4937-48 (2015).
Read more (PubMed: 26490646) »