Anti myc antibody

The ARF6-Myc, HA-RGA and HA-YFP proteins were synthesized by TNT T7 Quick Coupled in vitro transcription/translation system (Promega). The ARF6-Myc proteins were pre-incubated with anti-Myc antibody (Cell Signaling Technology)-bound protein A-Dynabeads (Life Technology) for 2 hr. After removing unbound ARF6-Myc proteins, the HA-RGA or HA-YFP proteins were incubated with the ARF6-Myc-bound Dynabeads for 1 hr in PBSN buffer (PBS buffer + 0.1% NP-40). The beads were washed three times with the PBSN buffer and the pulled-down proteins were analyzed by immunoblots using anti-HA antibody (Roche) and anti-Myc antibody (Cell Signaling Technology).

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).

Full-length human FAM46A/FAM46C with a FLAG-tag and BCCIPα/BCCIPβ with a Myc-tag were cloned into the pRK5 vector (556104, BD PharMingen). The plasmids were transfected into HEK293T cells with Lipofectamine 3000 (L3000001, Invitrogen) according to the manufacturer’s instruction. Cells were harvested 36 hours after transfection and lysed in the lysis buffer containing 50 mM tris (pH 8.0), 150 mM NaCl, 0.1% NP-40, 1 mM DTT, and protease inhibitor (78442, Sigma-Aldrich). Lysates were cleared by centrifugation at 15,000 rpm for 10 min at 4°C. anti-FLAG beads (A2220, Sigma-Aldrich) were added to the supernatant, and the mixture was rotated at 4°C for 1 hour. The beads were washed with a lysis buffer three times. Proteins remaining on the beads were resolved with 4 to 20% gradient SDS-PAGE (XP04205BOX, Invitrogen) and detected by Western blot using anti-FLAG (F7425, Sigma-Aldrich) and anti-Myc antibodies (2278, Cell Signaling Technology).