Rabbit anti human egfr

In brief, cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblotted with antibodies against EGFR and mAb806 epitope. The primary antibodies used included rabbit anti-human EGFR (#4267, Cell Signalling, Danvers, MA, USA) at 1:1000 dilution, mouse anti-human EGFR-806:mAb806 (Ludwig Institute for Cancer Research (LICR), Olivia-Newton John Cancer Research Institute (ONJCRI)) at 3 mg/mL and mouse anti-human actin (#3700, Cell Signalling, Danvers, MA, USA) at 1:1000 dilution. The secondary antibodies included goat anti-rabbit Ig (A2554, Sigma-Aldrich, St. Louis, MO, USA) at 1:3000 dilution and goat anti-mouse Ig (A6154, Sigma-Aldrich, St. Louis, MO, USA) at 1:3000 dilution.

For immunofluorescence staining, the cultured cells were fixed for 10 min in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and incubated overnight at 4°C with rabbit anti-human EGFR (#4267s; Cell Signaling Technology) and mouse anti-human CD44 (sc-18849; Santa Cruz Biotechnology, Inc.). Subsequently, the sections were rinsed and incubated with the secondary antibody conjugated to the fluorescent dye Alexa Fluor^® 488 and Alexa Fluor^® 594 (both from Jackson ImmunoResearch, West Grove, PA, USA). The sections were rinsed and incubated with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Life Technologies, Grand Island, NY, USA). A confocal laser scanning microscope (SP5; Leica, Wetzlar, Germany) was used to visualize the immunofluoresence staining.

Western blotting was performed as reported previously [46 (link)]. The antibodies used were as follows: rabbit anti-human NECTIN4 (1:1000, Abcam; ab192033), rabbit anti-human Akt (1:1000, #9272), rabbit anti-human phospho-Akt (1:2000, #4060), rabbit anti-human ERK (1:1000, #9102), rabbit anti-human phospho-ERK (1:2000, #4370), rabbit anti-human MEK (1:1000, #9122), rabbit anti-human phospho-MEK (1:1000, #9154), rabbit anti-human EGFR (1:1000, #4267), rabbit anti-human β-actin (1:2000, #4970), and goat anti-rabbit IgG horseradish peroxidase-linked secondary antibody (1:10,000, #7074) (all from Cell Signaling Technologies, Danvers, MA, USA). Immunological bands were visualized with SuperSignal^TM West Pico Chemiluminescence Substrate (Thermo Fisher Scientific; 34580) and captured with the ChemiDoc^TM XRS Plus System (Bio-Rad Laboratories; 1708265J1PC). The signals of bands were measured with Image Lab Software (Bio-Rad Laboratories).