Laemli sample buffer

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).

Semi-confluent HEKa cells were serum-starved for 3 h and then stimulated with 10 µg/ml DRGN-1 or HKGS for 5–30 min at 37 °C. AG1478 (0.2 µM final) was added 10 min prior to DRGN-1 or HKGS treatment. Cells were lysed with 2× Laemli sample buffer (Bio-Rad) containing 5 mM sodium fluoride and 2 mM sodium vanadate. Cell lysates were separated on a 4–12 % SDS-PAGE gel and transferred to polyvinylidene difluoride membrane using an iBlot system (Invitrogen). The membranes were probed with antibodies specific to phospho-EGFR and EGFR (Cell Signaling). The membranes were then treated with horseradish peroxidase-conjugated donkey anti-rabbit IgG (Abcam) and developed by SuperSignal West Femto Chemiluminescent Substrate (Fisher Scientific) under GelDoc Imaging System (Bio-Rad).

Cells and homogenized tissue were mixed with RIPA buffer (Thermo Scientific, USA) with 100 × protease inhibitor cocktail (GeneDEPOT, USA), and concentrations were measured by BCA assay. Protein lysate plus 2 × Laemli sample buffer (Bio-Rad, USA) was boiled at 97 °C for 10 min, separated by SDS-PAGE, and transferred to nitrocellulose membranes (Bio-Rad, USA). Membranes were blocked in 5% skim milk/TBST and incubated with 1/3000 diluted primary antibodies and 1/10,000 diluted secondary antibodies. ECL reagent (Thermo Scientific, USA) was used to obtain fluorescence signals from the X-ray films. The antibodies used are listed in Additional file 1, Table S1.