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. 2014 Feb 5;4:3977. doi: 10.1038/srep03977

Figure 3. EGFR tyrosine kinase inhibitors prevent tyrosine phosphorylation of the receptor (a) and protein phosphorylation in downstream signaling pathways (b) in cancer cells exposed to compounds NSC 228155 or CN 009543V.

Figure 3

MDA MB468 cells serum-starved overnight were pre-incubated (or not) with 10 μM AG1478 or 2 μM PD 153035 for 90 min and then, where indicated, incubated with 100 μM NSC 228155 or CN 009543V or 150 ng/ml EGF or vehicle (0.2% DMSO) for 15 min.Proteins were blotted to nitrocellulose membrane and analyzed with biotinylated anti-pTyr P100, anti-pEGFR Y1068 and anti-EGFR (epitope in cytoplasmic region) antibodies. The phospho-specific antibodies were anti-phospho-Shc (Tyr239/240), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204), anti-phospho-c-CBL (Tyr774), and anti-Gab1 (Tyr627). The signal intensity of the α-tubulin band was quantitatively evaluated and used as the loading control of protein samples.