Novex 10 nu page gels

Cell lysates prepared by extraction in 6 M Urea, 25 mM Tris base, 2% SDS, 2% β-mercaptoethanol, and 5% glycerol were analyzed by SDS-PAGE (10 µg/lane) using Novex 10% Nu-PAGE gels (Invitrogen) and the MES buffer system. Immunoblot analyses were performed using the Novex XCell SureLock Mini-Cell system (Life Technologies) and nitrocellulose membranes (Invitrogen). Transferred proteins were detected using IRDye-labeled secondary antibodies and the Odyssey infrared imaging system (Li-COR Biosciences). The primary antibodies used were, phospho-Smad 1/5/8 (Cell Signaling Technology Cat# 9511, RRID:AB_331671) and Smad1 (Cell Signaling Technology Cat #9517, RRID:AB_10699149).Peptide antibodies specific to XSMOC-1∆EC (SDRDRDPQCNPHCTRPQHK) or XSMOC-1EC (GSFPPGKRPGSNPFSR) were produced in rabbits (Biomatik Corp.).

Cell lysates were prepared by extraction in 6M Urea, 25mM Tris base, 2% SDS; aliquots (10μg) were mixed with 1X LDS sample buffer (Invitrogen)/2% mercaptoethanol prior to analysis by SDS-PAGE using Novex 10% Nu-PAGE gels (Invitrogen) and the MES buffer system. Immunoblot analyses were performed using the Novex XCell SureLock^® Mini-Cell system (Life Technologies) and nitrocellulose membranes (Invitrogen). Transferred proteins were detected using IRDye-labeled secondary antibodies and the Odyssey infrared imaging system (Li-COR Biosciences). The primary antibodies used were phospho p44/42 MAPK (dpErk), p44/42 MAPK, phospho-MEK1/2, EGFR (Cell Signaling Technology #9101, 9107, 9121, and 2239), pEGFR (Y1172; Abcam^® #ab47364), mature EGF (EMD Millipore # PC08), and pro-EGF (R and D Systems #AF4289).