Phosphorylated ikk α

After 2 and 4 h of incubation of Ti substrates with THP-1-deprived macrophages in the presence of different concentrations of forsythiaside, total proteins were extracted from cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer containing 0.1% phenylmethane sulfonyl fluoride (PMSF). Lysates were centrifuged at 15,000×g for 15 min, and the supernatants were collected. Protein concentration was determined using the BCA assay. Then, each protein lysate was resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on 10% gels and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% skimmed milk in Tris-buffered saline–Tween 20 (TBST) solution for 1 h and then incubated with primary antibodies IKK-α, phosphorylated IKK-α, IκB-α, phosphorylated IκB-α, MyD88, and GAPDH (Cell Signaling Technology, USA) diluted in 1% (w/v) skimmed milk powder in TBST overnight at 4 °C. Membranes were washed and incubated with the appropriate secondary antibodies. Antibody reactivity was detected by exposure in the Taton imaging system.

Paraquat, maneb and taurine were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). RNAiso Plus and SYBR Premix Ex Taq^TM II were obtained from Takara Bio Inc. (Takara, Tokyo, Japan). The membrane protein extraction kit was obtained from Beyotime (Jiangsu, China). The following primary antibodies were used: TH (EMD Millipore Corporation, Billerica, MA, USA), α-synuclein (Abcam, Cambridge, MA, USA), Iba-1 (Wako Chemicals, Richmond, VA, USA), CD11b (AbD Serotec, Raleigh, NC, USA), phosphorylated p65, p65, phosphorylated IκBα, IκBα, phosphorylated IKKα (Cell Signaling Technology, Danvers, MA, USA), 4-HNE (Abcam, Cambridge, MA, USA), p47^phox (EMD Millipore, Temecula, CA, USA), gp91^phox (BD Transduction Laboratories, San Jose, CA, USA) and GAPDH (Abcam, Cambridge, MA, USA). The BCA Protein Assay Kit was purchased from Life Technologies (Waltham, MA USA). All other chemicals were of the highest grade commercially available.