Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with <t>RIPA</t> buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on <t>SDS-acrylamide</t> gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p
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