EGFR expression of the cells used in this study was measured by Western blotting and flow cytometry.
Whole-cell lysates were collected and Western blotting was performed as described previously18 (link). Primary antibodies and the titres used in this study were as follows: rabbit monoclonal anti-EGFR antibody (D38B1, #4267, Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000), rabbit monoclonal anti-β-Actin antibody (13E5, #5125, Cell Signaling; 1:5,000). β-Actin served as loading controls for whole-cell lysates.
EGFR expression on the cell surface was detected with flow cytometry by measuring the fluorointensity of dye-conjugated anti-EGFR antibody which binds to cell-surface EGFR with a minor modification from a previous report38 (link). In brief, cells were washed twice with PBS, and they were stained for 30 minutes on ice with AlexaFluor 647-conjugated anti-EGFR or control antibodies in 2% FBS in PBS. Then, cells were washed twice with PBS and analysed with a flow cytometer (BD LSRFortessa Analyzer; BD Biosciences, San Jose, CA, USA). Antibodies and the titres were as follows: AlexaFluor 647-conjugated monoclonal anti-EGFR antibody (D38B1, #5588, Cell Signaling; 1:50), rabbit IgG isotype control (Alexa Fluor 647 Conjugate) antibody (#3452, Cell Signaling; 1:50). Collected data were analysed using FlowJo X software (Tree Star, Inc., Ashland, OR, USA).
Whole-cell lysates were collected and Western blotting was performed as described previously18 (link). Primary antibodies and the titres used in this study were as follows: rabbit monoclonal anti-EGFR antibody (D38B1, #4267, Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000), rabbit monoclonal anti-β-Actin antibody (13E5, #5125, Cell Signaling; 1:5,000). β-Actin served as loading controls for whole-cell lysates.
EGFR expression on the cell surface was detected with flow cytometry by measuring the fluorointensity of dye-conjugated anti-EGFR antibody which binds to cell-surface EGFR with a minor modification from a previous report38 (link). In brief, cells were washed twice with PBS, and they were stained for 30 minutes on ice with AlexaFluor 647-conjugated anti-EGFR or control antibodies in 2% FBS in PBS. Then, cells were washed twice with PBS and analysed with a flow cytometer (BD LSRFortessa Analyzer; BD Biosciences, San Jose, CA, USA). Antibodies and the titres were as follows: AlexaFluor 647-conjugated monoclonal anti-EGFR antibody (D38B1, #5588, Cell Signaling; 1:50), rabbit IgG isotype control (Alexa Fluor 647 Conjugate) antibody (#3452, Cell Signaling; 1:50). Collected data were analysed using FlowJo X software (Tree Star, Inc., Ashland, OR, USA).
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