Anti ttyh1

U2OS cells transfected with siRNAs grown on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature and were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). The cells were blocked with 3% bovine serum albumin and then incubated overnight at 4 °C with anti-TTYH1 (1:200; Cusabio technology LLC, Houston, TX, USA) antibody. After rinsing in cold PBS, the cells were treated with DyLight 488-conjugated secondary antibodies (1:500; Jackson Labs, Harbor, ME, USA) for 1 h at room temperature. The cells were washed, mounted, and observed under an A1 confocal microscope (Nikon, Minato-ku, Tokyo, Japan).

For western blotting, HEK293T, SNU-601, and TTYH1/2 double-knockout cells were lysed with lysis buffer (50 mM HEPES, 0.1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF, and 0.1% SDS containing protease inhibitor cocktail, pH 7.4). Total protein (15 μg/lane) was subjected to SDS-PAGE (8–12% gels) and transferred to PVDF membranes. The membranes were blocked using 5% non-fat milk, and then blotted with the appropriate antibodies: Anti-LRRC8A (Cell Signaling Technology, Inc., Danvers, MA, USA, #24979), anti-TTYH1 (CUSABIO technology LLC, Houston, TX, USA, CSB-PA867139LA01HU), and anti-actin (Sigma, St. Louis, MO, USA, A5441). The membranes were then washed and incubated with HRP-conjugated goat anti-mouse (Jackson ImmunoResearch, #115-035-166), goat anti-rabbit (Jackson ImmunoResearch, #111-035-144), or rabbit anti-goat IgG (Jackson ImmunoResearch Lab, #305-035-045), followed by washing and detection of immunoreactivity using ECL (Thermo, #34095).

U2OS cells transfected with siRNAs were extracted using RIPA butter (iNtRON Biotechnology Inc., Seongnam, Korea). Protein concentrations in the cells were determined using a bicinchoninic acid assay reagent kit (Pierce Chemical Co., Dallas, TX, USA) following the manufacturer’s instructions. The protein from each sample was separated by electrophoresis in 10% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk in TBST (Tris-buffered saline, 0.1% Tween 20) and incubated overnight at 4 °C with anti-TTYH1 (1:200; Cusabio technology LLC, Houston, TX, USA), anti-MMP2 (1:1000; Cell signaling technology, Danvers, MA, USA), anti-MMP9 (1:1000; Cell signaling), anti-N-cadherin (1:1000; Cell signaling), or anti-Actin (1:1000; Sigma Aldrich, St. Louis, MO, USA) antibodies. Then, the membranes were incubated with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The intensity of the bands was quantified by densitometry (ImageJ, Bethesda, MD, USA) and normalized to the corresponding actin bands. All experiments were performed at least three times.