Phospho erk thr202 thr204

BALB/cASlac-nu/nu nude mice bearing PDECX1T0326 tumors were orally administered with theliatinib (15 mg/kg) or gefitinib (20 mg/kg). After 8 h, the animals were sacrificed and tumors were harvested. The tumors were homogenized in cold lysis buffer (Cell Signaling Technology, 9803) containing 1 mM PMSF (BIO BASIC INC., PB0425) and after centrifuging the lysates, the protein supernatant (containing 100 μg protein) were mixed with 5× SDS loading buffer and boiled at 100°C for 10 minutes and SDS-PAGE was performed (5% stacking gel at 80V for 20 minutes, then changed to 120V on 10% separating gel for 1h). Proteins were then transferred to nitrocellulose membranes (0.35A for 90 minutes), and incubated with the following antibodies individually: Phospho-EGFR (Tyr1068) (Invitrogen, 44788G), EGFR (Cell Signaling Technology, 2232), phospho-AKT (Cell Signaling Technology, 4060), AKT (Cell Signaling Technology, 9272), phospho-ERK (Thr202/Thr204) (Cell Signaling Technology, 4370), ERK (Cell Signaling Technology, 4695). After washing with the 1X TBST, the blots were incubated with the secondary IRDye 800-conjugated secondary antibody (LI-COR, 926-32211) and detected with chemiluminescence system.

Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco BRL (Carlsbad, CA, USA). OEA, kojic acid, MK886, L-DOPA, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), α-melanocyte-stimulating hormone (α-MSH), PD98059 and PTU were purchased from Sigma-Aldrich Co. (St Louis, MO, USA). Antibodies against MC1R (1:1000, Cell Signaling Technology, Boston, USA), MITF (1:1000, Cell Signaling Technology), TRP-1 (1:1000, Cell Signaling Technology), Tyrosinase (1:1000, Cell Signaling Technology), PPARα (1:1000, Abcam, Cambridge, UK), total-ERK (1:1000, Cell Signaling Technology), phospho-ERK (Thr202/Thr204) (1:1000, Cell Signaling Technology), total-Akt (1:1000, Cell Signaling Technology), phospho-Akt (Ser473) (1:1000, Cell Signaling Technology), total-CREB (1:1000, Cell Signaling Technology), phospho-CREB (Ser133) (1:1000, Cell Signaling Technology), total-p38 (1:1000, Cell Signaling Technology), phospho-p38 (1:1000, Cell Signaling Technology) and mouse monoclonal anti-β-actin (1:10000, Sigma) were obtained from the indicated manufacturers. OEA, kojic acid, MK886, PD98059 and PTU were dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration was less than 0.05%. L-DOPA was dissolved in phosphate buffered saline (PBS).