Kctd11

Total protein samples were extracted quantitatively from the lysis buffer (Pierce, Rockford, IL). The reagent or bound protein was eluted from protein G PLUS‐agarose (Santa Cruz Biotechnology), separated by SDS‐PAGE, transferred onto polyvinylidene fluoride membranes (PVDF; Millipore, Billerica, MA, USA), incubated with antibodies overnight at 4℃, followed by addition of oxidase‐conjugated anti‐mouse or anti‐rabbit IgG (Santa Cruz Biotechnology) at 37℃ for washing and incubation for 2 h. Target protein expression levels were detected using an ECL kit (Thermo Fisher Scientific) and a bioimaging system (Bio‐Rad). WB analysis and IP were performed with the following antibodies: Myc‐tag, LATS1, YAP, P‐YAP, CTGF, β‐catenin, C‐myc, CyclinD1, MMP7, ZO‐1, N‐cadherin, E‐cadherin, vimentin, Snail, Slug, active β‐catenin, P‐β‐catenin (Cell Signaling Technology Inc. 1 μg/IP, 1:1000/WB); GAPDH (Sigma, 1:1000/WB); KCTD11 (Sigma, 1 μg/IP,1:1000/WB); LaminB1 (Abcam, 1:1,000/WB); and Tubulin (Abcam,1:1,000/WB).

The cells were fixed with 4% polyoxymethylene, permeabilized with Triton X‐100, blocked with normal goat serum at 37℃ for 1 h and incubated with primary antibody (KCTD11: 1:100, Sigma, β‐catenin, YAP: 1:100, CST) overnight at 4℃. Next, they were incubated with fluorescein isothiocyanate‐conjugated (FITC) or tetramethylrhodamine isothiocyanate‐conjugated (TRITC) secondary antibody at 37℃ for 2 h. Cell nuclei were stained with 4,6‐diamidino‐2‐phenylindole (DAPI). Cell images were acquired and analysed using an inverted Nikon TE300 microscope (Nikon Co., Ltd.) or a radiant 2,000 laser scanning confocal microscope (Oberkosen Carl Zeiss).