5.

Sp1 and Sp3 exert opposite effect on TβRII expression. A: HAC were trans-fected overnight with pEVR2-Sp1 or pCMV-Sp3 expression vectors (or with insertless plasmid as controls). Thereafter, media were replaced with DMEM + 2% FCS for 24 hrs in the absence or in the presence of IL-1β (1 ng/ml). Sp1 and Sp3 over-expression were checked by Western blot, and TβRII mRNA levels were analysed and expressed as relative expression versus GAPDH. B: HAC were nucleo-fected with Sp1 or Sp3 siRNA oligonu-cleotides. Thereafter, the medium was replaced with DMEM + 10% FCS for 24 hrs in the presence or in the absence of IL-1β (1 ng/ml). Then, Sp1 and Sp3 protein levels were defined by Western blot. Total RNA was also extracted, and real-time RT-PCR analysis was performed. Histograms represent the relative Sp1, Sp3 or TβRII mRNA levels versus GAPDH.