Poly l glutamic acid l tyrosine

The EGFR and c-Src kinase assays were performed in 96-well plates (NuncMaxisorp)
coated with PGT (poly L-glutamic acid L-tyrosine, 4:1, Sigma Aldrich, MO, USA)
and incubated at 37°C for 48h prior to using. PGT served as the substrate
to be phosphorylated by EGFR (Enzo Life Sciences Inc, NY, USA, Signal Chem,
Richmond, Canada) and c-Src (Enzo Life Sciences Inc, NY, USA, Signal Chem,
Richmond, Canada) in the presence of ATP (50 μM). A dose range of drugs
(I-VII, gefitinib or dasatinib) was added to compete with ATP
to bind and inhibit the ATP-binding site in the kinase domain of EGFR or c-Src.
To each well, 15 ng of EGFR (20 μg/ml) or 6 ng of c-Src (0.1
μg/μl) were added. The phosphorylated substrate was detected using
an HRP-conjugated anti-phosphotyrosine antibody (Santa Cruz Biotechnology, CA).
The signal was developed by the addition of 3, 3’, 5,
5’-tetramethylbenzidine peroxidase substrate (Kierkegaard and Perry
Laboratories, Gaithersburg, MD) and the colorimetric reaction was monitored at
450 nm using a microplate reader ELx808 (BioTek Instruments). The
IC₅₀ values were calculated using GraphPad Prism 6.0
(GraphPadSoftware, Inc., San Diego, CA). Each experiment was carried out at
least three times, in duplicate.

The EGFR kinase assays was performed in 96-well plate (NuncMaxisorp) coated with PGT (poly L-glutamic acid L-tyrosine, 4:1, Sigma Aldrich, MO, USA) and incubated at 37°C for 48 h prior to use. PGT is the substrate, which is phosphorylated by EGFR (Enzo Life Sciences Inc, NY, USA, Signal Chem, Richmond, Canada) in the presence of ATP (50 μM). A dose range of drugs (1×10^-7-10 μM, gefitinib or MR30) was added to compete with ATP to bind and inhibit the ATP-binding site of the EGFR kinase domain. To each well, 15 ng of EGFR (20 μg/ml) was added. The phosphorylated substrate was detected using an HRP-conjugated anti-phosphotyrosine antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The signal was developed by the addition of 3, 3’, 5, 5’-tetramethylbenzidine peroxidase substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD, USA) and the colorimetric reaction was monitored at 450 nm using a microplate reader ELx808 (BioTek Instruments) [57 (link)]. The IC₅₀ values were calculated using GraphPad Prism 6.0 (GraphPadSoftware, Inc., SanDiego, CA, USA). Each experiment was carried out twice in duplicate.