4.3. Western Blot Analysis of Cell Lysates

Gefitinib-sensitive and -resistant models were treated with EGFR TKI, FASN inhibitor, or the combination of both drugs for 72 h. Afterwards, attached and floating cells were harvested and lysed in ice-cold lysis buffer (Cell Signaling Technology Inc., MA, USA) containing 100 µg/mL phenylmethylsulfonylfluoride (PMSF; Sigma) by vortexing every 5 min for 30 min. Protein concentration was determined by the Lowry method (DC Protein Assay, Bio-Rad Laboratories, Inc.). Equal amounts of protein were heated in lithium dodecyl sulfate (LDS) sample buffer with a sample reducing agent (Invitrogen, CA, USA) for 10 min at 70 °C, separated by SDS-PAGE and transferred to nitrocellulose membranes (ThermoFisher Scientific Inc., MA, USA). Membranes were incubated for 1 h at room temperature in blocking buffer (5% skim milk powder in Tris-buffered saline (TBS)) 0.05% Tween (TBS-T) and overnight at 4 °C with the following primary antibodies diluted in blocking buffer: FASN (Cell Signaling Technology Inc.; #3180), p-STAT3^Tyr705 (Cell Signaling Technology Inc.; #9131), p-EGFR^Tyr1068 (Cell Signaling Technology Inc.; #2234), PARP (Cell Signaling Technology Inc.; #9542), EGFR (Cell Signaling Technology Inc.; #2232), STAT3 (Cell Signaling Technology Inc.; #4904), and GAPDH (Proteintech, Manchester, UK; #60004-1-IG). Specific horseradish peroxidase (HRP)-conjugated secondary antibodies were incubated for 1 h at room temperature. The immune complexes were detected using chemiluminescent HRP substrate Clarity^TM Western ECL Substrate (Bio-Rad Laboratories, Inc.) or West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific Inc.) in a Bio-Rad ChemiDoc^TM MP Imaging System (Bio-Rad Laboratories, Inc.). Western blot analyses were repeated at least three times and representative results are shown.