Anti-EGFR (phospho Y1068) antibody [EP774Y] (ab40815)

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). 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).

Blocking/Diluting buffer 5% NFDM/TBST

Formaldehyde-fixed, paraffin-embedded mouse e17 embryo head (Developing tooth) tissue stained for EGFR (phospho Y1068) using ab40815 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 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.

Blocking buffer: 5% NFDM/TBST
Dilution buffer: 5% NFDM/TBST

ab40815 showing positive staining in Cervical carcinoma tissue.

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

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