Sc 8303

Whole cell lysate protein was extracted by using RIPA Lysis Buffer (Santa Cruz) and sonicated with Branson Snifier 150. The concentration of extracted protein was measured based on Bradford dye-binding method by using Bio-Rad Protein Assay Reagent and Bio-Rad SmartSpecTM 3000 spectrophotometer. Protein samples were fractionated by size on NuPAGE 4–12% Bis-Tris Gel (Novex) by electrophoresis and then transferred to Nitrocellulose Membrane (Protran, Whatman). The probed primary antibodies were detected by using Horseradish Peroxidise (HRP)-conjugated secondary antibodies and the Enhanced Chemiluminescent (ECL) detection system (GE Health). Primary antibodies: α-SMA (A5228, Sigma-Aldrich, 1:1000); SM22α (ab14106, Abcam, 1:1000); Calponin (EP798Y, Abcam, 1:1000); FSP-1 (ab27957, Abcam, 1:500); SOX2 (ab59776, Abcam, 1:500); OCT4 (sc-5279, Santa Cruz, 1:500); KLF4 (sc-20691, Santa Cruz, 1:500); CD144 (ab33168, Abcam, 1:500); CD31 (252253, ABBIOTEC, 1:500); Claudin 5 (352588, life technologies, 1:500); E-Cadherin (3195 P, Cell Signaling, 1:500); SNAI1 (3879 P, Cell Signaling, 1:1000); N-Cadherin (ab12221, Abcam, 1:300); HES5 (Ab5708, Millipore, 1:400); JAG1 (sc8303, Santa Cruz, 1:300); GAPDH (ab8245, Abcam, 1:2000). Secondary antibodies: anti-rabbit (P0217, DAKO, 1:3000); anti-mouse (P0260, DAKO, 1:3000).

Proteins were extracted from cells or frozen tissue; the samples were run on SDS polyacrylamide gels of 8–15% depending on molecular weight of protein of interest and transferred on to nitrocellulose membrane. Following transfer the membranes were probed with primary antibodies for K-Ras (OP24, Calbiochem, EMD Millipore, MA, USA), GAPDH (Ma3374, Millipore), Jag 1 (sc-8303, Santa Cruz, CA, USA), pErK (4370, Cell Signaling, MA, USA), α-SMA (Ab5694, Abcam), Active Ras-GTP Monoclonal Antibody (26909, NewEast Bioscience) and subsequently probed with the appropriate secondary anti-mouse IgG or anti-rabbit IgG. Densitometry was performed using ImageJ software provided by NIH.