Emt antibody sampler kit

Cultured cells were lysed with CelLytic M Cell Lysis Reagent (Sigma; C2978) containing a protease inhibitor cocktail (Sigma; S8830). Protein concentration was quantified using a Pierce Bicinchonic Acid Assay Kit (Life Technologies). Western blotting was performed as described previously, using E-cadherin, vimentin and ZEB1 antibodies from the EMT Antibody Sampler Kit (Cell Signaling; 9782) and β-actin antibody (Sigma; A5316).

Proteins were extracted from cultured cells using cell lysis buffer (Cell Signaling, Beverly, MA, USA). Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA), which were blocked in 5% skim milk in TBST. Membranes were then incubated with primary antibody and HRP-conjugated secondary antibodies [anti-ALDH1A1, anti-phospho-FAK (Tyr397), anti-FAK, anti-survivin, anti-β-catenin, anti-Myc antibodies, and EMT antibody sampler kit (Cell Signaling); anti-IGFBP1, anti-IGFBP1, anti-CD44, and anti-CD166 antibodies (Abcam, Cambridge, UK), anti-CD133 antibody (MyBioSource, San Diego, California, USA)]. The specific complexes were detected using a SuperSignal West Pico kit (Thermo Scientific, Rockford, IL, USA). Data were quantified and analyzed using a GS-800 calibrated densitometer (Bio-Rad, Hercules, CA, USA). Relative band intensity values were calculated by normalizing the experimental absolute intensity to that of the corresponding β-actin band as a loading control. Additionally, five CLM samples in paraffin blocks were randomly selected from the 18 patients of the initial RNA-Seq to examine the degree of contamination of normal liver tissues by IHC using Heppar-1 and CK-7 (DAKO, Carpinteria, CA, USA) monoclonal antibodies to hepatocytes and bile duct cells, respectively, as previously described [17 (link)] (S2 Fig).

Western blotting analysis was performed as described previously [30 (link)]. Primary antibodies against LSH and α-tubulin were purchased from Santa Cruz Biotechnology, and primary antibody against GINS4 was purchased from GeneTex. EMT Antibody Sampler Kit and primary antibodies against histone H3 were purchased from Cell Signaling Technology, and primary antibody against β-actin was purchased from Sigma-Aldrich (St. Louis, MO).