Glass bottom cell culture dishes

The hBMEC were seeded onto 15 mm Glass Bottom Cell Culture Dishes (Corning, NY, USA) and treated with 10 μg/ml BifA or BifA variants for 2 hours. Cells were then fixed with 4% paraformaldehyde followed by 0.1% Triton X-100 permeabilization buffer and blocked with 5% BSA in PBS-Tween. Mouse polyclonal anti-BifA antibody, Alexa 488-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA, USA), rabbit anti-Moesin antibody (Abcam, Cambridge, MA, USA) and Alexa 594-conjugated goat anti-rabbit antibody (Jackson Immunoresearch, West Grove, PA, USA) were used at 1:2000 in PBS containing 1% BSA. Primary antibodies were incubated for 2 hours and secondary antibody for 1.5 hours at room temperature. 4,6- Diamidino-2-phenylindole‎ (DAPI) was used to detect cell nuclei. Plates were washed three times with Phosphate buffered saline with Tween-20 (PBST) with shaking to wash out unbound antibodies. Images were obtained on a laser scanning confocal microscope (LSCM) (ZEISS, Japan).

Cells were seeded on glass-bottom cell culture dishes with a 20 mm-diameter (Corning) and grown to 30% confluence. Cells were fixed with 4% paraformaldehyde for 15 min, followed by washing with PBS three times. Then, cells were permeated by 0.5% Triton X-100 and blocked by 5% BSA for both 30 min at room temperature. Cells were incubated with rabbit anti-AEG-1 antibody (Proteintech, 1:1000), mouse anti-GSK-3β antibody (Cell Signaling Technology, 1:1000), and rabbit anti-γH2AX (Cell Signaling Technology, 1:400), diluted using 1% FBS, at 4 °C overnight. Goat anti-rabbit Cy3-conjugated, FITC-conjugated secondary antibody, and goat anti-mouse Alexa Fluor Plus 488-conjugated secondary antibody (Invitrogen) were added and incubated for 1 h at 37 °C away from light. And then, Antifade Mounting Medium with DAPI (Beyotime Biotechnology) was supplemented for nuclear staining. The samples were imaged using Leica TCS SP5 confocal microscope.