Anti myc tag

Total protein was extracted from cells using RIPA buffer containing 10% protease inhibitor cocktail (Sigma, St. Louis, MO). Protein concentration was determined using Bradford (Bio-Rad Laboratories, Hercules, CA), and 40 μg of each sample was fractionated by 10–12% SDS-PAGE and blotted onto a nitrocellulose membrane (Hybond-ECL; Amersham Biosciences, Little Chalfont, UK). Non-specific binding sites were blocked with 5% non-fat dry milk in PBS−0.1% Tween-20. The following primary antibodies were used: anti-FAM83F (N17) (sc-102517), anti-p-ERK1/2 (sc-7393), anti-ERK1(sc-94), anti-BRAF (sc-166), anti-RAF-1 (sc-133), anti-Vimentin (sc-32322), anti-TTF-1 (sc-13040), anti PAX-8 (sc-81353), anti-β-actin (sc-47778) (Santa Cruz, Santa Cruz, CA, USA), anti-Myc-tag (TA100010) (OriGene Technologies), and anti-LIN28B (Cell Signaling). The anti-Nis antibody was kindly donated by Dr. Sissy Jhiang.
Immunoexpression was detected with horseradish peroxidase-conjugated secondary antibodies (GeHealthcare) and developed with luminol and p-cumaric acid (Sigma) reagents in the presence of H₂O₂. Chemoluminescence emission was visualized in an ImageQuant LAS4000 imaging system (GE Healthcare, Little Chalfont, UK).

For immunoprecipitation, PCCL3-FAM83F and Nthy-ori-FAM83F cells were lysed in RIPA buffer containing 10% protease inhibitor cocktail and incubated with agarose-protein A/G beads (Santa Cruz) for pre-clearing. Next, cell lysates were incubated overnight at 4°C under agitation with 20 μL of the complex of agarose-protein A/G beads + 1 μg of one of the following antibodies: anti-Myc tag (OriGene Technologies), anti-BRAF (Santa Cruz), anti-RAF1 (Santa Cruz), or anti-HuR (Invitrogen). After the incubation, beads were pelleted at 1,000 × g at 4°C and washed in RIPA buffer. Beads were resuspended in 1 × Western Blotting loading buffer and denaturated at 95°C for 5 min before loading into 10% SDS-PAGE. Immunodetection was performed as already described in WB section.