Monoclonal anti β actin

Denatured protein lysates were resolved by 4–12 % NuPAGE gels (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose membranes (Schleicher & Schuell, Dachen, Germany). The membranes were incubated with anti-5-LO (BD, Franklin Lakes, NJ); anti-p-Akt (Ser473), anti-pan-Akt (Cell signaling, Danvers, MA); or monoclonal anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature or overnight at 4 °C. Membranes were then washed (4 times) with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL) for 1 h. Immunoreactive proteins were visualized by developing them with Lumi-light western blotting substrate (Roche Diagnostics GmbH, Mannheim, Germany), followed by exposure in a LAS-3000 (Fuji Film Co., Tokyo, Japan) according to the manufacturer’s instructions. This was followed by quantitation of specific bands with the Multi Gauge software (Fuji Film Co., Tokyo, Japan).

Hippocampal proteins were extracted from each group using lysis buffer (Beyotime, China) and phosphatase inhibitor cocktail (Thermo Fisher Science, USA). Protein samples were mixed with loading buffer, heated at 100° C for 5 min, then separated on 10% SDS-PAGE and transferred to PVDF membranes. Then, blots were blocked with 5% nonfat dried milk for 2 h at room temperature. Monoclonal anti-Ppp1c (diluted 1 : 2000, molecular weight: 39 kDa, Abcam, USA), monoclonal anti-Arsa (diluted 1 : 2000, molecular weight: 54 kDa, Abcam, USA), anti-Tau (phospho S262 + T263) (diluted 1 : 2000, molecular weight: 79 kDa, Abcam, USA), anti- total Tau (diluted 1 : 2000, molecular weight: 52 kDa, Abcam, USA) or monoclonal anti-β-actin (diluted 1 : 1000, molecular weight: 42 kDa, Santa Cruz, USA) were used as primary antibodies. Anti-rabbit or anti-mouse IgG HRPs (diluted 1: 3000, Thermo Fisher Scientific, USA) were used as secondary antibodies. After washing in TBST, the membranes were exposed to chemiluminescence reagents using an electronic chemiluminescence kit on a phosphor imager (Pierce Biotechnology, USA) and analyzed for optical density based on Java (ImageJ) software (National Institutes of Health, Bethesda, MD, USA).

Human gingival fibroblasts, isolated as previously described, were seeded in 12-well culture plates (2×10⁵ cells/well) and incubated for 5, 10, 15 and 30 min with or without IL-1β (100 pg/ml) or TNF-α (50 ng/ml) (R&D systems, Inc, Minneapolis, USA). Cells were collected, lysed and protein concentration was measured as described above (ELISA section). The lysates were resolved by 10% TRIS-HCl polyacrylamide gel electrophoresis and proteins were transferred onto a nitrocellulose membrane. Protein detection was performed using primary polyclonal rabbit anti-human Iκα antibodies (clone C-21, cat. no. sc-371, Santa Cruz Biotechnology, CA, USA) or monoclonal anti-β-actin (cat. no. A5441, Sigma Aldrich, St. Louis, MO, USA) followed by secondary horseradish peroxidase-conjugated anti-rabbit (cat. no. P0448, Dako, Glostrup, Denmark) and anti-mouse (cat. no. 31340, Thermo Scientific, Rockford, IL, USA) antibodies respectively. The immunoreactive proteins were visualized using the ECL Plus Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) and analyzed by ImageQuant LAS 4000 imager.