Western Blot Analysis of Claudin-1, Occludin, Afadin, ZO-1, ZO-2, NF-κB, and X-Linked Inhibitor of Apoptosis Protein (XIAP)

For sampling of cellular extract for western blot analysis, 5 × 10⁵ cells were seeded 1 week before irradiation. Forty-eight hours after irradiation and both in presence/absence of PBMC in the basolateral compartment, cells were lysed with Cell Lysis buffer (Cell Signaling Technology) following the manufacturer instruction and cellular extracts were stored at −20°C. Total protein quantification was performed with BCA method (Abcam) according to manufacturer instruction.

Proteins were mixed with Laemli Sample Buffer (BioRad) additionated with β-mercaptoethanol (BioRad) and heated at 95°C for 5 min, then centrifuged few seconds at 10,000 g. The same amount of proteins underwent electrophoresis in 4–20% precast gels (BioRad), and subsequently proteins were transferred on PVDF membranes (BioRad). After the blocking step with non-fat dry milk 5% in PBS 0.2% Tween-20, membranes were incubated overnight with primary antibodies: anti-claudin-1, anti-ZO-1, anti-ZO-2, anti-afadin (Cell Signaling Technology), anti-occludin (Millipore), anti-NF-κB (Epitomics), and anti-XIAP (Abcam). Samples were then incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibody (Amersham). Films were obtained after visualization with enhanced chemoluminescent kit (BioRad), and scanned with Gel Doc EZ Imager (BioRad). Finally, bands were quantified with Image Lab 4.0 software (BioRad).