Protein lysis and western blot

Cells were washed with phosphate-buffered saline (PBS) and lysed in 1× lysis buffer (Cell Signaling Technology, USA) for 10 min. The whole protein of cells was centrifuged at 12,000 rpm for 10 min. The protein concentration of the supernatant was measured by the BCA reagents (Thermo, Waltham, MA, USA). Equal amount of proteins was resolved by SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were masked using 5% skim milk in TBS-T for 1 h at room temperature (RT) and incubated sequentially with primary antibody to ZO-1 (1:200) and beta actin (1:5000) (Abcam, Cambridge, MA, USA) and alpha tubulin (1:5000) (Sigma-Aldrich, St Louis, MO, USA). Then, membranes were treated for 1 h with HRP-conjugated secondary antibody (1:5000). Antibody binding was detected using ECL reagents and chemiluminescence (GE healthcare).