Anti p erk1 2 t202 y204 4370

HEK293, SKBR3, U2OS, MCF7 and A549 cells were grown in DMEM supplemented with 10% FBS, penicillin (50 units/mL), and streptomycin (50 μg/mL) in a 37 °C incubator containing 5% CO₂. KYSE-520 and H358 cells were grown in RPMI-1640 supplemented with 10% FBS, penicillin (50 units/mL), and streptomycin (50 μg/mL) in a 37 °C incubator containing 5% CO₂. For western-blot analysis, protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated overnight at 4 °C with corresponding primary antibodies diluted in 5% BSA in phosphate-buffered saline Tween (PBST). SuperSignal™ West Pico PLUS (PI34580; Thermo Scientific) was used to visualize the antibodies in the bioanalytical imaging system (Azure Biosystems c500). Anti-p-ERK1/2 (T202/Y204) (4370), and anti-ERK1/2 (4696) were purchased from Cell Signaling Technology. Anti-GAPDH (sc-365062), anti-SHP2 (sc-7384), anti-SHP1 (sc-7289), and anti-Actin (sc-8432) antibodies were purchased from Santa Cruz. Anti-PTP1B (ab244207) antibody was purchased from Abcam. For colony formation assay, a total of approximately 200–500 cells were seeded to each well in a 12-well plate. The wells were then treated with DMSO, compound P9 and 3 at 37 °C for 3 weeks, gently washed, and stained with crystal violet.

For dose-response analysis, subconfluent cells were incubated in serum-deprived medium for 24 h, stimulated with increasing ligand concentrations for 24 h at 37°C and solubilized in radioimmune precipitation (RIPA) buffer; for time-course experiments, subconfluent cells were treated with IGF-I for the indicated time points.
To evaluate IGF-I - dependent activation of DDR1, cells were serum-starved for 24 h, and then stimulated with IGF-I (50 nM) for 24 h.
Cell lysates were subjected to western blot analysis, as previously described [49 (link)]. The following antibodies were used: anti-DDR1 (C-20, sc-532, Santa Cruz Biotechnology), anti-phospho(p)-IR/p-IGF-IR (Y1150/Y1151, 3024, Cell Signaling), anti-IGF-IR (9750, Cell Signaling), anti-p-AKT (Ser473, 9271, Cell Signaling), anti-AKT(9272, Cell Signaling), anti-p-ERK1/2 (T202/Y204, 4370, Cell Signaling), anti-ERK1/2 (9102, Cell Signaling), anti-p-p70 (Thr421/Ser424, 9204, Cell Signaling); anti-p70 (C-18, 230 Santa Cruz Biotechnology); anti-α-tubulin (15246, Abcam); anti-actin (Sigma).

HEK293, SKBR3, U2OS, MCF7 and A549 cells were grown in DMEM supplemented with 10% FBS, penicillin (50 units/mL), and streptomycin (50 μg/mL) in a 37 °C incubator containing 5% CO₂. KYSE-520 and H358 cells were grown in RPMI-1640 supplemented with 10% FBS, penicillin (50 units/mL), and streptomycin (50 μg/mL) in a 37 °C incubator containing 5% CO₂. For western-blot analysis, protein samples were separated by SDS-PAGE, transferred to nitrocellulose membranes, and incubated overnight at 4 °C with corresponding primary antibodies diluted in 5% BSA in phosphate-buffered saline Tween (PBST). SuperSignal™ West Pico PLUS (PI34580; Thermo Scientific) was used to visualize the antibodies in the bioanalytical imaging system (Azure Biosystems c500). Anti-p-ERK1/2 (T202/Y204) (4370), and anti-ERK1/2 (4696) were purchased from Cell Signaling Technology. Anti-GAPDH (sc-365062), anti-SHP2 (sc-7384), anti-SHP1 (sc-7289), and anti-Actin (sc-8432) antibodies were purchased from Santa Cruz. Anti-PTP1B (ab244207) antibody was purchased from Abcam. For colony formation assay, a total of approximately 200–500 cells were seeded to each well in a 12-well plate. The wells were then treated with DMSO, compound P9 and 3 at 37 °C for 3 weeks, gently washed, and stained with crystal violet.