Anti 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).

The lysates from HL-1 cells were harvested with lysis buffer (25 mmol/L Tris-HCl-pH 7.6, 0.3 mol/L NaCl, 1.5 mmol/L MgCl₂, 0.2 mmol/L EDTA, 0.5% Nonidet P-40, and 0.5 mmol/L dithiothreitol) and immunoprecipitated with anti-ubiquitinylated protein antibodies (Chemicon, Millipore, Temecula, CA, USA) for 2 h at 4°C. Following incubation with 50 μL of Protein A-Sepharose for 1 h at 4°C, beads were collected, washed three times, and resuspended in SDS sample buffer. The immunocomplexes were resolved by SDS-PAGE and analyzed by Western blot with anti-XIAP and anti-SMA (Abcam, Cambridge, MA, USA).

Protein was extracted using protein extraction solution (PRO-PREP, iNtRON Biotechnology, Kyungki-Do, Korea). The protein, 20 µg, was electrophoresed on a 10% polyacrylamide gel, and transferred to a nitrocellulose membrane (Atoh, Tokyo, Japan). The membranes were reacted overnight at 4°C with the primary antibodies. The primary antibodies applied were anti-ATP5A (Proteintech), anti-8-OHdG (ABBIOTEC, San Diego, CA), anti-XIAP (Abcam), anti-cleaved-caspase-3 (Abcam), or anti-caspase-9 (Proteintech) at a concentration of 0.5, 2.5, 1.0, 1.0, or 1.0 µg/ml. After the incubation with peroxidase-labeled goat anti-mouse or anti-rabbit IgG antibody (Dako Cytomation) for 1 hour at room temperature and vigorous washing, the nitrocellulose membrane was incubated with Chemiluminescence Luminol Reagent (Immuno Star LD, Wako, Tokyo, Japan) and photographed digitally using ImageQuant LAS 4000 mini (GE healthcare Japan Co, Tokyo, Japan). Immunoblot using anti-actin monoclonal antibody (Sigma Chemical Co. St. Louis, MO) was used for standardization. Intensity was measured using the Multi Gauge v3.1 (Fujifilm, Tokyo, Japan). Experiments were repeated at least three times.

Total protein was isolated from cell lysates by using RIPA buffer and quantified by the BCA protein assay kit (Beyotime, Shanghai, China). Proteins were resolved on 10% SDS-PAGE and then transferred to PVDF (Bio-Rad) membranes. After blocking, the membranes were incubated with primary antibodies at 4°C overnight. Then, the membranes were incubated with secondary anti-rabbit antibody (Abcam; 1:5,000) at room temperature for 1 h. Membranes were scanned by using an Odyssey Imaging System and analyzed with Odyssey v2.0 software (LICOR Biosciences, Lincoln, NE, USA). The primary antibodies used in this study are as follows: anti-NCAPG2 (Abcam, Cambridge, MA, USA; 1:1,000), anti-Bax (Abcam; 1:1,000), anti-XIAP (Abcam; 1:1,000), anti-activated caspase 9 (Abcam; 1:1,000), anti-E-cadherin (Abcam; 1:1,000), anti-N-cadherin (Abcam; 1:1,000), anti-Vimentin (Abcam; 1:1,000), and anti-β-actin (Abcam; 1:1,000). β-actin was used as an internal control.