Heme oxygenase 1 ho 1

The spinal cord tissues were taken out from liquid nitrogen, lysed in RIPA lysis buffer, containing protease inhibitor, for 30 min and centrifuged to obtain the supernatant which was used to determine the protein concentration. After SDS-PAGE, the proteins were transferred onto a membrane, sealed and incubated overnight at 4°C with antibodies against NF-κB (Abcam, USA), p-NF-κB (Abcam, USA), B-cell lymphoma-2 (Bcl-2) (Abcam, USA), Bcl-2-associated X protein (Bax) (Abcam, USA), phosphatidylinositol 3-kinase (PI3K) (Abcam, USA), protein kinase B (Akt) (Abcam, USA), phosphorylated (p)-Akt (Abcam, USA), heme oxygenase-1 (HO-1) (Abcam, USA), Nrf2 (Abcam, USA), trithorax-1 (TRX-1) (Abcam, USA), Raf-1 (Abcam, USA), MEK (Abcam, USA), ERK (Abcam, USA), p-MEK (Abcam, USA), p-ERK (Abcam, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, USA) (diluted at 1:1000). Next, the PVDF membrane (Sigma, USA) was cleansed in TBST and incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 2 h. Finally, the color of the proteins was developed using an ECL kit and gel imaging system, and the absorbance analyzed by Image Tools.

Western blotting was performed as previously described.19, 20 The following primary antibodies were used: β‐Actin, ICAM, IL‐1β, Sirt1 (Santa Cruz Biotechnology, Dallas, TX, USA); PGC‐1α, CPT‐1B, phospho‐GSK‐3β (p‐GSK‐3β), total‐GSK‐3β (t‐GSK‐3β), phospho‐AMPKα (p‐AMPKα), total‐AMPKα (t‐AMPKα) (Cell Signaling Technology, Beverly, MA, USA); 3‐nitrotyrosine (3‐NT, Millipore Corp., Temecula, CA); Anti‐4 hydroxynonenal (4‐HNE, Alpha Diagnostic Int.) and PPARα, CD36, Laminin, FN, TNF‐α, SOD2 and heme oxygenase‐1 (HO‐1) (Abcam, Cambridge, MA, USA). The corresponding secondary antibody and β‐Actin were employed as an internal control.

Equal amounts of proteins (40 μg total protein or 80 μg nuclear protein per lane) were loaded onto a 12.5% gradient Tris-HCl SDS polyacrylamide gel and then transferred to a PVDF membrane. Nonspecific binding to the membrane was blocked for 1 h at room temperature with 5% nonfat milk in 1 × TBS, followed by incubation with primary antibodies against total Akt (rabbit monoclonal 1 : 1000; Cell Signaling Technology, Danvers, MA), p-Akt (Ser473, rabbit monoclonal 1 : 2,000; Cell Signaling Technology), Nrf2 (mouse monoclonal 1 : 1000; Abcam Inc., Cambridge, MA, USA), heme oxygenase 1 (HO-1, mouse monoclonal 1 : 500; Abcam), β-actin (mouse monoclonal polyclonal 1 : 1,000; Abcam), or Histone H3 (mouse monoclonal polyclonal 1 : 500; Abcam) overnight at 4°C. After washing with TBST three times, membranes were incubated with horseradish peroxidase-conjugated rabbit or goat secondary antibody (1 : 10000 dilution; Kang Chen Biotechnology, Guangzhou, China) for 1 h at room temperature, followed by three washes for 10 min each. Blots were developed using enhanced chemiluminescent reagents (Thermo Fisher Scientific, Pittsburgh, PA, USA) and target band density was scanned using a LAS-3000 detection system. Image J software was used to analyze band intensities. The results were normalized to the protein levels of β-actin or Histone H3.

Samples from the CPu or from cultured astrocytes were solubilized in buffer containing 1% NP40, 0.1% sodium dodecyl sulfate, and 0.2% deoxycholate and were subjected to western blotting with the following antibodies: tyrosine hydroxylase (TH; EMD Millipore, Billerica, MA, USA), glial fibrillary acidic protein (GFAP; Dako, Glostrup, Denmark), GRP78 (StressGen, Victoria, British Columbia, Canada), heme oxygenase-1 (HO-1; Abcam, Cambridge, UK), and β-actin (Sigma). Primary antibody binding was visualized using the ECL system (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).

Nuclear and cytoplasmic proteins in the tissue homogenate were fractionated using NE-PER^TM Nuclear and Cytoplasmic Protein Extraction Kit (Thermo Fisher Scientific., Rockford, IL, USA), as previously described [22 (link)]. After quantification and denaturation, the proteins were electrophoretically separated on a sodium dodecyl sulfate polyacrylamide gels, and the separated bands were transferred onto polyvinylidene fluoride membranes (Merck Millipore Corp., Billerica, MA, USA). The primary antibodies used in this study were directed to COX-2 (Cell Signaling Technology, Danvers, MA, USA), heme oxygenase-1 (HO-1; Abcam, Cambridge, UK), or β-actin (Santa Cruz Biotechnology, Dallas, TX, USA). Appropriate secondary antibodies, HRP-conjugated, were used for each primary antibody. Protein bands were developed, digitalized, and densitometrically analyzed using Image Studio Lite version 5.2 (LI-COR Biotechnology, Lincoln, NE, USA).