Gapdh

HaCaT cells were pretreated with 200 μg/mL RBE and exposed to 100 mJ/cm² UVB radiation. The protein derived from the treatment was isolated using 1x cell lysis buffer (Cell Signaling), and the concentration was measured using the Bradford Protein Assay Kit (AMRESCO). Protein lysates were evaluated with Western blot analyses as previously described [20 (link), 21 (link)]. Western blot analysis was performed using the specific antibodies: PARP, caspase-3 (DAKO), catalase (Bioss), Cu/ZnSOD (ABBIOTEC), GAPDH, MnSOD, Nrf2, HO-1, β-actin, phos-p38, p38, c-Jun, NFκBp65, and NFκBp50 (Santa Cruz). The levels of GAPDH or β-actin were used as the internal loading control. Densitometric analyses of scanned images were performed using GeneTools software (Syngene, UK).

Total protein was extracted from donor heart tissues (Qiagen DNeasy kit; Qiagen) according to the manufacturer's instructions. Proteins (20 μg of total protein) were subsequently separated by SDSPAGE, and then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk in Tris‐buffered saline with Tween‐20 (TBST) at room temperature for 2 hours. Then, the proteins were incubated overnight with rabbit antimouse IDO (Abcam; 1:200) at 4°C. The membranes were then incubated with HRP‐conjugated goat anti‐rabbit second antibody (Abbiotec; 1:1000) for 1 hour at room temperature. After washing the membrane thrice with TBST, the proteins were visualized using an ECL detection system (G: BOX, Syngene). GAPDH (Abbiotec; 1:1000) was used as the internal control. The density of each band was determined using the corresponding GAPDH value. Bands were scanned and quantitated by densitometry using Quantity one imaging software.