N acetyl s farnesyl l cysteine

Lipopolysaccharide (LPS, Escherichia coli O55:B5) was purchased from Calbiochem. Antibodies for inducible nitric oxide synthase (iNOS) and β-actin were purchased from BD Transduction Lab and Novus Biologicals, respectively. Cyclooxygenase-2 (COX-2) and glial fibrillary acidic protein (GFAP) were purchased from Abcam. Total STAT3 and phospho-STAT3 (p-STAT3) were purchased from Cell Signaling Technology. ELISA kits for TNFα and IL-6 were purchased from Invitrogen. 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and N-acetyl-S-farnesyl-L-cysteine (AFC) were purchased from Sigma-Aldrich. Poly-ornithine was purchased from BD Biosciences. Papain and DNase I were purchased from Worthington Biochemical. Fetal bovine serum was purchased from HyClone and heat-inactivated. Culture media and penicillin/streptomycin were purchased from Gibco, and other common chemicals were from Sigma-Aldrich, unless stated otherwise.

A non-radioactive colorimetric continuous enzyme kit (#786-430, G-Biosciences, San Jose, CA, USA) was used to measure the enzyme activity of ICMT. We added 5 μL of SAM methyltransferase assay buffer in the blank group, 5 µL of sample transfected with ICMT or ICMT E167A in the sample group, and 5 µL of ICMT substrate (N-acetyl- S-farnesyl-L-cysteine, Sigma). We added 10 µL of ICMT inhibitor (CyM) as needed. Then, 100 µL of SAM Methyltransferase Master Mix (SAM Methyltransferase Assay Buffer, SAM Enzyme Mix, SAM Colorimetric Mix, S-adenosylhomocysteine) was added to all wells. We used a kinetic spectrophotometer at an absorbance wavelength of 510 nm and 37 °C once every 30 s for 30 min.