Western blotting analysis using phospho-EGFR (Y1068) antibody (Cat#61211). Total cell lysates (30 μg) from various cell lines were loaded and separated by SDS-PAGE. The blot was incubated with phospho-EGFR (Y1068) antibody (Cat#61211, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using NaQ™ ECL Substrate Kit (Cat#716).
Western blotting analysis using phospho-EGFR (Y1068) antibody (Cat#61211). Phospho-EGFR (Y1068) expression in wild type (WT) and EGFR shRNA knockdown (KD) HepG2 cells with 20 μg of total cell lysates. Hsp90 α serves as a loading control. The blot was incubated with phospho-EGFR (Y1068) antibody (Cat#61211, 1:5,000) and HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000) respectively. Image was developed using NaQ™ ECL Substrate Kit (Cat#716).
Immunohistochemistry was performed on paraffin-embedded human lung adenocarcinoma using phospho-EGFR (Y1068) antibody (Cat#61211, 1:200). Antigen retrieval was done in sodium citrate buffer (pH 6.0). DAB was used for detection, with hematoxylin counterstaining. Images were acquired using a Nikon Ci-L Plus microscope (40× objective). Scale bar: 25 μm.
Immunocytochemical staining of HepG2 cells using Phospho-EGFR (Y1068) antibody (Cat#61211, 1:1,000), Top panel: wild-type (WT); Bottom panal: Phospho-EGFR (Y1068) shRNA knockdown (KD). Nuclei were stained blue with DAPI;Phospho-EGFR (Y1068) was stained magenta with Alexa Fluor® 647. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 0.5% Triton X-100 in 1x PBS. Scale bar: 20 μm.
Validation of EGFR knockdown using flow cytometry. Wild-type(WT, Blue) and knockdown(KD, Green) HepG2 cells were stained with Phospho-EGFR (Y1068) antibody (Cat#61211, 1:2,000) and analyzed using BD flow cytometer. Fixative solution: 4% Paraformaldehyde in 1x PBS; Permeabilization solution: 100% Methanol, chill on -20℃ before use.
