Rabbit anti α catenin

Briefly, cells were washed with phosphate buffered saline (PBS) before harvesting with lysis buffer (Sangon Biotech, Co., Ltd., Shanghai, China). Protein was separated by SDS‐PAGE and transferred to a Polyvinylidene Fluoride (PVDF) membrane. Non‐specific binding was blocked with Tris Buffered Saline Tween (TBST) containing 5% (w/v) non‐fat dried milk. The membrane was then incubated with rabbit anti‐ATF3 (1:1000; Abcam, Cambridge, MA, USA), rabbit anti‐α‐catenin (1:50000; Abcam), rabbit anti‐E‐cadherin (1:10000; Abcam), rabbit anti‐Vimentin (1:2000; Abcam), rabbit anti‐Fibronectin (1:1000; Abcam), rabbit anti‐p53 (1:1000; Abcam), rabbit anti‐MDM2 (1:1000; Abcam), rabbit anti‐Bax (1:1500; Abcam), rabbit anti‐Slug (1:1000; Abcam), rabbit anti‐Snail1 (1:1000; Abcam), rabbit anti‐Twist (0.5 μg/mL, Abcam) and rabbit anti‐GAPDH (1:500; Abcam) at 4°C overnight. Next, membranes were incubated overnight with an Horseradish Peroxidase (HRP)‐conjugated goat anti‐rabbit IgG (1:10000; Abcam) for 1 hour at 37°C. The membranes were developed with an Emitter‐Coupled Logic (ECL) chemiluminescence detection kit and quantitated using ImageJ software. GAPDH served as a loading control.

For IF staining, hCMEC-D3 grown under normoxia/OGDR conditions were co-cultured with and without hPMSCs. The cells were washed with wash buffer (PBS+MgCl₂+CaCl₂+protease inhibitor), and then fixed in ice-cold 4% paraformaldehyde for 10 min on ice, and permeabilized (0.5% Triton X-100/PBS, 5 min, 25⁰C). Cells were blocked with 5% BSA/5% goat serum for 1 h at 25⁰C. Primary antibodies (rabbit anti-α-catenin (1:100, Abcam) and rabbit anti-VE-cadherin (1:100, Cell Signaling) were diluted in wash buffer and incubated with cells overnight at 4 °C. Cells were next incubated with fluorescently conjugated secondary antibody (AlexaFluor 488 goat anti-rabbit; Life Technologies; USA) for 1 h and rinsed twice. Hoechst (Thermo Scientific; USA) was added to the cells for 5 min, washed, mounted on glass slides and images recorded using a Nikon video at 20X magnification. Images were processed with Image-J software.

After desired treatments, cells collected in Laemmli buffer (Bio-Rad; USA) containing 10% 2-mercaptoethanol. The cells were scraped and sonicated at power of 50% for 15 sec, boiled at 95 °C for 15 min. 20 μl of protein was separated via SDS-PAGE, then immunoblotted to PVDF and incubated at 4 °C with rabbit anti-ZO-1 (1:500), rabbit anti-α-claudin-1 (1:500), (Invitrogen), rabbit anti-occludin (1:1000), rabbit anti-α-catenin (1:1000,Abcam), rabbit anti-VE-cadherin (1:1000) and rabbit anti-β-tubulin (1:2000,Cell Signaling). Membranes were incubated with goat anti-rabbit IgG-HRP antibodies (1:2500, Sigma) for 2 h at 25 °C. Signal was detected using ChemiDocTM MP imaging system (Bio-Rad) and results analyzed with NIH Image-J software.