Inhibition of EGFR Phosphorylation in A431 Cells

The inhibition of EGFR phosphorylation was studied as described in the literature [13 (link)]. A431 cells (5 × 10⁵ cells/well) were seeded in 6-well plates and incubated for 24 h. Then, the medium was replaced with fresh medium without fetal bovine serum. The next day, cells were incubated in the presence of the compound at the following concentrations: 0.1 nM, 1 nM, 10 nM, 50 nM, 100 nM, 1 μΜ, and 10 μΜ for 2 h, and then EGF was added (20 ng/mL) and left for 5 min. Two control samples were included in the experiment containing cell cultures without the compound and with or without EGF as a positive and negative control, respectively. Cells were lysed with cell lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM, NaCl, 1 % Triton X-100, 0.25 % w/v sodium deoxycholate, 0.1 % w/v, sodium dodecyl sulfate, protease, and phosphatase inhibitors with EDTA). Ten micrograms of total protein for each sample were loaded onto polyacrylamide gel (8%) for electrophoresis. Subsequently, the proteins were separated by electrophoresis and transferred to a nitrocellulose membrane. Next, the membrane was washed three times in TBS-T buffer (tris-buffered saline and Tween 20), blocked for one hour in TBS-T with 5% milk (1% fat), and incubated for two hours minimum in a phosphotyrosine antibody (p-Tyr 100, mouse mAb) diluted 1/1000. Afterward, the membrane was washed three times in TBS-T and incubated in an anti-mouse IgG (HRP Linked) antibody diluted 1/1000 for 1 h. Then, the membrane was rewashed three times in TBS-T. The detection was performed using a chemiluminescent detection system (ECL kit, SignalFire ECL Reagent, Cell Signaling Technology, Auburn, MA, USA) according to the manufacturer’s instructions. Protein band quantification was performed using Image-J software, and the percentage of EGFR phosphorylation inhibition was calculated using GraphPad Prism 9.0 software.