Anti lrrc8a antibody

The human GC cell lines MKN7, MKN45, MKN74, HGC27, and NUGC4 were obtained from the Riken Cell Bank (Tsukuba, Japan). These cell lines were cultured in RPMI-1640 (Nacalai Tesque, Kyoto, Japan) supplemented with 100 µg/ml of streptomycin, 100 U/ml of penicillin (Nacalai Tesque), and 10% fetal bovine serum (FBS) (Sigma-Aldrich Japan, Tokyo, Japan) in a humidified incubator at 37 °C in 5% CO 2 . The rabbit polyclonal anti-LRRC8A antibody used in the immunohistochemical (IHC) analysis was purchased from Abcam (ab-157489, Cambridge, MA, UK). The mouse monoclonal anti-LRRC8A antibody used in western blotting was purchased from Sigma-Aldrich (SAB1412855 St. Louis, MO, USA). A mouse monoclonal anti-beta-actin (ACTB) antibody was purchased from Sigma-Aldrich (A5441). Rabbit polyclonal anti-SAPK/JNK, anti-phospho-SAPK/JUN, and anti-phospho-p53 (Thr81) antibodies, a mouse monoclonal anti-p53 (1C12) antibody, and a rabbit anti-p21 (Waf1/ Cip1) antibody were purchased from Cell Signaling Technology (anti-SAPK/JNK; #9252/anti-phospho-SAPK/JNK; #9251/anti-phospho-p53 (Thr81); #2676/anti-p53 (1C12); #2524/anti-p21 (Waf1/Cip1); #2947, Beverly, MA, USA). Anti-horseradish peroxidase (HRP)-linked anti-mouse and anti-rabbit secondary antibodies were obtained from Cell Signaling Technology (anti-mouse; 7076S/anti-rabbit; 7074S).

NUGC4 cells were directly seeded and cultured on glass coverslips, followed by fixation with 4% paraformaldehyde. They permeabilized using 0.1% Polyethylene Glycol Monop-isooctylphenyl Ether (Triton X-100) (Nacalai Tesque). Cells were blocked with 1% bovine serum albumin and stained using the rabbit polyclonal anti-LRRC8A antibody (Abcam), rhodamine phalloidin (Setareh Biotech, Eugene, OR, USA), and DAPI (Fujifilm Wako, Osaka, Japan). The distribution of LRRC8A in NUGC4 cells was observed under a fluorescence-microscope (BZ-X810, Keyence, Osaka, Japan).