Flow Cytometry analysis of A431 (Human epidermoid carcinoma epithelial cell) treated with 200 ng/ml EGF for 15 minutes cells labeling EGFR with purified ab40815 at 1:800 dilution (1 µg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) secondary antibody was used at 1:2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). Untreated A431 cells (Green).

All lanes : Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815) at 1/1000 dilution (Purified)

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes whole cell lysates
Lane 3 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes. Then the membrane was incubated with phosphatase

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 170 kDa
Observed band size: 175 kDa why is the actual band size different from the predicted?

This antibody detects high background.

Immunocytochemistry/ Immunofluorescence analysis of A431 (Human epidermoid carcinoma epithelial cell) treated with 100 ng/ml EGF for 10 minutes cells labeling EGFR with purified ab40815 at 1:500 dilution (1.8 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1:200 (2.5 µg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1:1000 (2 µg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815) at 1/1000 dilution (purified)

Lane 1 : A431 (Human epidermoid carcinoma epithelial cell) whole cell lysates
Lane 2 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes whole cell lysates
Lane 3 : A431 (Human epidermoid carcinoma epithelial cell) treated with 10 ng/ml EGF for 30 minutes. Then the membrane was incubated with phosphatase

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 170 kDa

This antibody detects high background.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling EGFR with purified ab40815 at 1:500 dilution (1.75 µg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Total EGFR (green) subcellular localization with and without caerulein-induced pancreatitis as determined by immunohistochemistry and confocal imaging. The nuclei were identified with DAPI stain (blue). Scale bars = 50 μm. Calreticulin and E-cadherin (red) served as markers for the endoplasmic reticulum and plasma membrane, respectively, and were used to quantify EGFR subcellular location. Pancreatitis was induced with the 1-day protocol of 8 hourly caerulein injections in 6–8 week old wild-type (wt) and 3-week old AGR2^-/- (ko) mice.

For immunofluorescence, antigen retrieval was performed in a pressure cooker set to 118°C. The slides were incubated in antigen unmasking solution (DAKO) for 3 min followed by equilibration at room temperature for 1 hr. The slides were then placed in 5% serum blocking solution (goat, horse, or rabbit serum as appropriate) for 30 min to block nonspecific binding of antibody to the tissue. The sections were incubated with primary antibody diluted in 2% serum overnight at 4°C. The respective secondary antibodies were used at predetermined dilutions. Immunofluorescence slides were mounted with media containing DAPI stain (Vectashield, Vector Laboratories).

WD-PBEC cultures were infected with RSV clinical isolate BT2a and stained for EGFR (red) and RSV F (RSV F, green) expression.

For WD-PBECs, pediatric bronchial epithelial cells (PBEC) were obtained, via written parental consent, from bronchial brushings of children undergoing elective surgery at the Royal Belfast Hospital for Sick Children, and the procedures were approved by the Office of Research Ethics Committees Northern Ireland. PBEC were expanded in collagen-coated flasks using airway epithelial cell media and supplements (Lonza), then seeded onto transwell inserts (Corning), and then air-liquid interface (ALI) cultures were initiated and maintained 21 days in order to establish well-differentiated (WD)-PBECs. Paraformaldehyde-fixed and permeabilized WD-PBEC were stained for RSV F protein expression and were stained with anti-phospho-(p)-EGFR (Abcam, ab40815, unpurified). WD-PBEC cultures were infected with RSV subgroup A clinical isolate BT2a. Fluorescent images were obtained with a SP5 confocal DMI 6000 inverted microscope (Leica).

Formaldehyde-fixed, paraffin-embedded mouse e17 embryo head (Developing tooth) tissue stained for EGFR (phospho Y1068) using ab40815 (unpurified) at 1/1000 dilution in immunohistochemical analysis, followed by Goat anti Rabbit IgG (Biotin) at 1/300 dilution.

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Dot blot analysis of EGFR (pY1068) peptide (Lane 1), SMAD5 (unmodified) peptide labelling EGFR (pY1068) with ab40815 (unpurified) at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody at a dilution of 1/2500.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 3 minutes.

Immunohistochemical staining of untreated (A) and Phosphatase- treated (B) paraffin-embedded breast adenocarcinoma tissue using ab40815 (unpurified).

Immunofluorescent staining of untreated (C) and Phosphatase- treated (D) A431 cells using ab40815 (unpurified).

ab40815 (unpurified) showing positive staining in Papillary carcinoma of thyroid gland tissue.

ab40815 (unpurified) showing positive staining in Glioma tissue.

ab40815 (unpurified) showing positive staining in Cervical carcinoma tissue.

Equilibrium disassociation constant (K_D)
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Formaldehyde-fixed, paraffin-embedded human prostate cancer tissue stained for EGFR (phospho Y1068) using ab40815 (unpurified) at 1/200 dilution in immunohistochemical analysis, followed by Goat anti Rabbit IgG (Biotin).

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