Fam84a

Cells treated differently were fixed in formaldehyde for 20 min and permeabilized with Triton X‐100 for 5 min at room temperature. Then blocked for 1 h with 2% BSA, cells were incubated with primary antibodies β‐catenin (1 : 100, CST) and FAM84A (1 : 200, Abcam) at 4 °C overnight. Afterward, cells were incubated with secondary antibodies (Cy3™ goat anti‐rabbit IgG, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h at room temperature. The cell nuclei were stained with DAPI for 5 min. Photographs were taken using a confocal microscope (Nikon).

Immunohistochemistry staining was performed on tumor sections from PTC patients and nude mice. The sections were treated with primary antibodies against FAM84A (1 : 200, Abcam) or Ki‐67 (1 : 500, Abcam), which was used to evaluate the proliferation, and then incubated with secondary antibodies. The images of all samples were observed with a microscope.

Protein was extracted and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Darmstadt, Germany). After being blocked in 5% nonfat milk, the membranes were incubated with the following primary antibodies at 4 °C overnight: FAM84A (Abcam, 1 : 1500 Cambridge, MA, USA), E‐cadherin (CST, 1 : 1000), N‐cadherin (CST, 1 : 1000), vimentin (CST, 1 : 1000), CDK‐4 (CST, 1 : 1000), CDK‐6 (CST, 1 : 1000), Bcl‐2 (CST, 1 : 1000), Bax (CST, 1 : 1000), and β‐catenin (CST, 1 : 1000), followed by a brief wash and incubation with secondary antibody for 2 h at room temperature. Anti‐GAPDH and anti‐histone H3 antibodies were purchased from Beyotime (Shanghai, China) and used as loading control. The expression of protein was visualized by the enhanced chemiluminescence detection system.