Fig. 5.
Interaction between RBPs and MITF and effect of pLTA on the mRNA stability of melanogenic proteins. The 1 × 105 B16F10 cells (n = 3) were incubated with α-MSH for 48 h in the presence or absence of pLTA. RNA EMSA was conducted with nuclear extracts containing protein and 10 fmol of biotinylated probe. Anti-hnRNP A1 and anti-HuR antibodies were added to the reaction. The mixture was separated by electrophoresis and detected by immunoblot analysis with anti-hnRNP A1 or anti-HuR antibody. Arrows indicate the position of the hnRNP A1/HuR band (A). Immunoprecipitation assays were performed with anti-MITF antibody for identification of hnRNP A1 or HuR-MITF complexes. Precipitated proteins were analyzed by immunoblot assays using anti-hnRNP A1 or anti-HuR antibody (B). In order to clarify the influence of pLTA, the protein levels of hnRNP A1 and HuR after pLTA treatment were measured by Western blot analysis. To verify the amount of loaded protein, the blots were probed with anti-β-actin (C).