Nanog 14295 1 ap

DCC-2036 (Rebastinib) was purchased from Selleck (Houston, TX). The compound was dissolved in DMSO at a stock concentration of 20 mM and stored at −20 °C. DCC-2036 (0 μM) means DMSO treatment with equivalent volume. Recombinant Human GAS6 (C-Fc) #C12W was purchased from Novoprotein (Suzhou, Jiangsu, China). Recombinant Mouse GAS6 Protein (His Tag) #58026-M08H was purchased from Sino Biological (Shanghai, China). Cycloheximide and MG132 were purchased from Sigma-Aldrich (St Louis, MO, USA). The following antibodies were used for immunoblotting: phospho-AXL (Y702) #5724, phospho-AKT (S473) #4060, AKT #4691,phospho-GSK3β (S9) #5558, GSK3β #12456, β-actin #4970, purchased from Cell Signaling Technology (Beverley, MA); ALDH1A1 #15910-1-AP, OCT4 #11263-1-AP, KLF4 #11880-1-AP, SOX2 #11064-1-AP, KLF5 #21017-1-AP, NANOG #14295-1-AP, AXL #13196-1-AP, MET #25869-1-AP, FGF-BP1 #25006-1-AP purchased from Proteintech (Wuhan, Hubei, China); NF-κB (P65) #ab16502, purchased from Abcam; FGF-BP1 #AF1593 purchased from R&D; phospho-KLF5 (S303) #AF7042, phospho-AXL (Tyr702) # AF8523 purchased from Affinity (Changzhou, Jiangsu, China). APC anti-mouse CD24 #138506 purchased from Biolegend (San Diego, CA, USA), PE Rat anti-mouse CD44 #553134, PE Mouse Anti-Human CD24 #555428, APC Mouse Anti-Human CD44 #559942 purchased from BD Pharmingen (Franklin Lakes, New Jersey, USA).

Proteins were collected from CRC cells and tissues using RIPA lysis buffer (EpiZyme, China), and protein concentrations were detected using a BCA kit (Beyotime, China). 30 μg of protein per group was loaded on a 10% SDS-PAGE gel, transferred to a PVDF membrane (Millipore, USA) and then closed with 5% skimmed milk. The blots were cut prior to hybridization with specific antibodies according to the protein molecular weight during blotting. Thereby, decreasing the amount of incubation solution used during the antibody incubation step, contributing to reduce the use of the costly antibodies. The membranes were incubated overnight at 4 °C in primary antibodies: SLC12A2 (13,884–1-AP, Proteintech, China), C-myc (5605S, CST, USA), Nanog (14,295–1-AP, Proteintech, China), CD44 (37259S, CST, USA), GAPDH (Servicebio, China). Finally, the cells were incubated with the corresponding secondary antibodies for 2 h and the bands were detected by enhanced chemiluminescence reagent (EpiZyme, China).