Anti rabbit igg

3, 4’-DHF was purchased from Indofine Chemical Inc. (Hillsborough, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, St. Louis, MO, USA). The ROS scavenger, NAC, and the MEK kinase inhibitor, U0126, were obtained from Calbiochem (San Diego, CA, USA). Dexamethasone, β-glycerophosphate, ascorbic acid, alkaline phosphatase kits, and alizarin red s were purchased from Sigma-Aldrich (St. Louis, MO, USA). The reagent for measuring intracellular ROS, 2’, 7’-dichlorodi-hydrofluorescein diacetate (H₂DCFDA), was obtained from Molecular Probes (Eugene, OR, USA). Primary antibodies for β-actin, phospho-ERK, ERK, phospho-AKT, and AKT were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), and the peroxidase conjugated secondary antibodies, anti-mouse IgG, anti-rabbit IgG, anti-goat IgG, were obtained from Amersham Bioscience (Piscataway, NJ, USA).

Non-irradiated or irradiated cells were trypsinized and counted immediately after irradiation, and the same number of non-irradiated and irradiated cells was plated onto dishes containing fresh media. Two days after irradiation, cells were lysed with RIPA lysis buffer containing PMSF and sodium orthovanadate (Santa Cruz Biotechnology, Dalla, TX, USA) and then used for the western blotting.
For the proteasome inhibitor treatment, two patterns of schedule were used: 1) 10 nM of epoxomicin (proteasome inhibitor; Peptide Institute Inc., Osaka, Japan) was added to the culture media immediately before cell irradiation, and the cells were cultured for 48 h in a 5% CO₂ incubator at 37°C and then lysed, or 2) 10 nM of epoxomicin was added to the culture media 48 h after irradiation, and the cells were cultured in a 5% CO₂ incubator at 37°C for 24 h. The cells were then lysed with RIPA lysis buffer and used for the western blotting.
Immunoblotting was performed as previously described (37 (link)). Primary antibodies for human Fra-1 (Santa Cruz Biotechnology) and GAPDH (Trevigen, Bristol, UK) with horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG (Amersham Biosciences, Buckinghamshire, UK) were used for this study. Protein bands were detected by enhanced chemiluminescence and imaged using a Lumino image analyzer (LAS4000; Fujifilm, Tokyo, Japan).

Immunoblotting was carried out as described previously.14 (link) All antibodies were used at dilutions of 1:1000, with the exception of anti-β-actin antibody (1:5000). For immunodetection, secondary anti-rabbit IgG or anti-mouse IgG (Amersham, Piscataway, NJ, USA) antibodies were used at a dilution of 1:5000. Antibody binding was detected using the ECL system (Amersham).