Anti p67phox

We observed changes in the protein expression of NADPH oxidase subunits after acupuncture via western blot analysis. The hippocampus samples were homogenized and centrifuged for 10 min at 4 °C. Protein concentration in the supernatants was determined using a Micro BCA protein assay (Thermo Scientific). The protein samples were resolved by SDS-PAGE gels (Applygen) and transferred to PVDF membranes. After being blocked with 5% skim milk at room temperature for 1 h and washed four times for 5 min each time in TBST, blots were subsequently incubated with anti-gp91phox (1:5000; Epitomics), anti-p47phox (1:200; Santa Cruz Biotechnology), or anti-p67phox (1:100; Santa Cruz Biotechnology) overnight at 4 °C. After being washed, the membranes were further incubated with a secondary antibody (1:10000; KBL) for 60 min. The average intensity values of protein bands were scanned and analysed by an infrared imaging system (Odyssey). β-actin or GAPDH was used as the control for gel loading and protein transfer.

For Western blot analysis, 100 μg of total kidney protein were separated on SDS-polyacrylamide minigels by electrophoresis (16 (link)). After transfer by electroelution to PVDF membranes (GE Healthcare Limited, Little Chalfont, UK), blots were blocked for 1 h with 5% non-fat milk in Tris-buffered saline solution. Blots were then incubated with primary antibodies for anti-eNOS (BD Bioscience, CA, USA); anti-iNOS (MyBiosource, CA, USA); anti-HO-1 (AssayDesigns, MI, USA); anti-MnSOD and anti-Nrf2 (Cayman Chemicals, MI, USA); anti-p47^phox and anti-p67^phox (Santa Cruz Biotechnology, CA, USA). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (anti-mouse or anti-rabbit, Sigma Chemical, MO, USA) and enhanced chemiluminescence detection (GE Healthcare Limited, Little Chalfont, UK). Kidney protein levels were further analyzed with a gel documentation system (Alliance 4.2; Uvitec, Cambridge, UK) and the software Image J for Windows (Image J-NIH Image). We used densitometry to quantitatively analyze the protein levels, normalizing the bands to β-actin expression (anti-β-actin, Sigma Chemical, MO, USA).

Total protein was prepared by homogenising hippocampal and post-hypothalamic tissues in lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor cocktail. The sample was subsequently incubated for 1 h at 4 °C. The protein extracts (20 μg/sample based on the Bicinchoninic Acid (BCA) protein assay, Pierce Chemical Co., Rockford, IL, USA) were resolved on a 6% polyacrylamide gel and transferred to a PVDF membrane (GE Healthcare, Buckinghamshire, UK). The membranes were incubated in the appropriate anti-P-Tau^T231 (ab151559), anti-P-Akt^S473 (4060, Cell Signaling Technology, Beverly, MA, USA), anti-P-GSK-3bS9 (05-643, EMD Millipore, Billerica, MA, USA), anti-P-GSK-3β^Y216 (ab75745) antibodies at 1:1000 in PBST with 5% BSA at 4 °C overnight, anti-amyloid precursor protein (ab12266), anti-gp91-phox (sc5827, Santa Cruz Biotechnology, Dallas, TX, USA), anti-Tau (ab80579), anti-Akt (9272), anti-p67-phox (sc7663), anti-GSK-3β (07-389), anti-p47-phox (sc14015), anti-p22-phox (sc11712), or anti-caspase 3 (9662) antibodies at 1:1000 in PBST with 5% BSA at room temperature (RT) for 1 h. The membranes were then incubated in an HRP-labelled goat anti-rabbit secondary antibody at 1:10,000. The membranes were developed using an ECL-Plus detection kit (GE Healthcare).