Electrochemiluminescence

Post-irradiation, at 0 h, 0.5 h, and 8 h, preparation of total cell lysates was performed using the ProtectJETTM Mammalian Cell Lysis Reagent from Fermentas (Lithuania) as per the protocol provided by the manufacturer. The membranes with the transferred protein were incubated with gentle agitation with following specific primary antibodies (1 : 1,000) at 4°C overnight: p-ATM (1 : 1000), p-DNA-PKcs (1 : 1000), Rad51 (1 : 1000), MMP10 (1 : 1000), and actin (1 : 1000) (all primary antibodies were from Abcam, USA). The secondary antibody (1 : 5000) were also from Abcam. Electrochemiluminescence (Santa Cruz Biotechnology Inc) was used to detect all the membranes.

Equal amounts of proteins were extracted from tissue and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After transferring them to nitrocellulose membranes (Perkin Elmer, Waltham, MA, USA), the proteins were incubated with primary antibodies against ALDH2, 4-HNE, HO-1 (Abcam, Cambridge, UK), Nrf2, (ABclonal, MA, USA), tubulin and GAPDH (Santa Cruz, TX, USA). Signals were detected with electrochemiluminescence (Santa Cruz, TX, USA) and quantified by densitometry. Data in the linear immunoreactive range were normalized to GAPDH or α-tubulin as a loading control.

Triton X-100 extracts (50 µg/lane) from colon samples were immunoblotted. Gelatin-agarose-purified PBS extracts were immunoblotted under non-reducing conditions. Cell-culture supernatants were concentrated using a vacuum centrifuge and volumetrically analyzed by immunoblotting.
Immunoblots were probed with anti-PPARγ (Cell Signaling), anti-MMP-9 (Abcam), anti-Lcn2 (Abcam), and anti-β-actin (Santa Cruz Biotechnology) antibodies. Immunodetection with an appropriate secondary peroxidase-conjugated antibody (DAKO) was followed by electrochemiluminescence (Santa Cruz Biotechnology). Quantification of protein bands was performed with the LabImage software. Fold changes were calculated using densitometry values for bands representing proteins of interest, normalized to densitometry values for β-actin bands of respective samples. Representative blots from at least two independent experiments are shown.