Kras 12063 1 ap

Total colon cancer cell lysates obtained from different experiments were separated using the SDS-PAGE using Mini-Protean III system (Bio-Rad, Taiwan) and transferred onto PVDF membranes using Trans-Blot Turbo Transfer System (Bio-Rad, Taiwan). The majority of the primary antibodies used in this study were listed. YAP1 (#4912), mTOR (#2983), and β-catenin (#9562) from Cell Signaling Techology (Taipei, Taiwan) and Kras (12063-1-AP), IL-6 (21865-1-AP), and IL-4 (16545-1-AP) from Protein Tech (Taipei, Taiwan). Secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Protein of interests were detected and visualized using ECL detection kit. Images were obtained and analyzed using UVP BioDoc-It system (Upland, CA, USA).

Dulbecco's Modified Eagle's Medium (DMEM), FBS, Penicillin/Streptomycin and Glutamine were all purchased from Life Technologies. BIRB796 (S1574), CB1954 (S7829), PLX-4072 (S1267), Vemurafenib (S1267) and ZSTK474 (S1072) were purchased from Selleckchem; Trametinib (HY-10999) was from Insight Biotechnologies; JNK inhibitor VIII (CAS 894804-07-0) was from Calbiochem; SCH772984 (S7101) was from Merck. Bovine serum albumin (BSA), DMSO and Doxycycline were purchased from Sigma. Vectashield (H-1200-10) was purchased from VectorLabs. The following antibodies were all purchased from Cell Signalling Technologies: p-S473 AKT (4060), Akt1 (2967), c-Jun (9165), p-T202/Y204 ERK1/2 (4370), ERK1/2 (9107), GFP (2955), p-S217/S221 MEK1/2 (9154), MEK1/2 (4694), p-T334 MAPKAPK2 (8753), MAPKAPK2 (3042). BRAF (3967S) was purchased from Santa Cruz. Tubulin (T9026) and β-actin (A5441) were purchased from Sigma and KRAS (12063-1-AP) was from ProteinTech. Horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad and the enhanced chemiluminescence (ECL) system from GE Healthcare was used for detection. The Dylight™680 and Dylight™800 conjugated secondary antibodies were from Cell Signalling Technology.