Enhanced chemiluminescence ecl western blotting detection system

Dulbecco's modified Eagle's medium (DMEM)/F-12 and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). 5, 8-Dihydroxy-4′,7-dimethoxyflavone (DDF, Figure 1) was kindly provided by the Natural Products Laboratory of Dr. Yann-Lii Leu (Chang Gung University, Taiwan). Hybond C membrane and enhanced chemiluminescence (ECL) Western blotting detection system were purchased from GE Healthcare Biosciences (Buckinghamshire, England, UK). Antibodies against phospho-p38 and phospho-Nrf2 were purchased from Cell Signaling (Danvers, MA). Actinomycin D (Act.D), cycloheximide (CHI), N-acetyl cysteine, SP600125, GSH, trolox, diphenyleneiodonium chloride (DPI), PD98059, and p38 inhibitor VIII (p38i VIII) were purchased from Biomol (Plymouth Meeting, PA). Anti-β-actin antibody was purchased from Biogenesis (Boumemouth, UK). Anti-GAPDH (#MCA-1D4) was obtained from EnCor Biotechnology (Gainesville, FL). Anti-HO-1 pAb and Glutathione (GSSG/GSH) detection kits were purchased from Enzo Life Sciences (Farmingdale, NY). CTGF antibody (sc-25440) was purchased from Santa Cruz (Santa Cruz, CA, USA). Mitotempol, dihydroethidium (DHE), and CM-H₂DCFDA were purchased from Molecular Probes (Eugene, OR, USA). SDS-PAGE reagents were purchased from MDBio Inc. (Taipei, Taiwan). Thrombin and other chemicals were purchased from Sigma (St. Louis, MO, USA).

For western blot analysis, sciatic nerves were grossly dissected into the small fragments, and then, the tissue lysates were made using TissueLyser LT (Qiagen) in modified RIPA buffer containing 1% Triton X-100 in Tris–EDTA solution. Proteins were centrifuged at 9000 × g for 10 min at 4 °C, and the supernatant was collected. The RIPA lysates (10–35 μg) were separated by SDS-PAGE, and then transferred onto a nitrocellulose membrane (Amersham Biosciences). After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST; pH. 7.2) for 1 h at room temperature, the membranes were incubated with primary antibodies (1: 500–2000) in TBST containing 1% nonfat dry milk overnight at 4 °C. After three washes with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. Chemiluminescence reactions were performed using an enhanced chemiluminescence (ECL) western blotting detection system (GE Healthcare), and then, the images were detected using a Luminogragh 3 and quantified with CS Analyzer 4 software (ATTO). Quantification was performed with the density analyzer in CS Analyzer 4, and the values were obtained from three independent experiments. The list of antibodies is shown in Table S3.