Total and active TGF-β1 and p-Smad2 levels increase transiently after acute CCl4 challenge in WT mice but not in PF4CreTgfb1f/fmice. (A) Total TGF-β1 levels in the plasma of WT and PF4CreTgfb1f/f mice at the indicated time points were measured with a modified ELISA protocol using DuoSet kits from R&D Systems. There was a transient increase in the total plasma TGF-β1 in WT mice (n = 7) but not in PF4CreTgfb1f/f mice at 6 hours (n = 9). (B) Both active and total TGF-β1 levels in liver exudates were measured by ELISA. Active TGF-β1 levels were higher in WT liver exudates than in PF4CreTgfb1f/f liver exudates at 6 hours (n = 5; P = .004). (C) p-Smad2 immunostaining showed that most of the phosphorylated signals were in the nuclei (red dots in the tiled pictures of the whole-liver section in the top panels and magnified injured areas in the bottom panels), showing lower levels of p-Smad2 in the injured liver areas of PF4CreTgfb1f/f vs WT mice at 3 days after CCl4 challenge. (D) Quantification of p-Smad2 intensity in the injured areas of the liver sections showed that PF4CreTgfb1f/f livers had fewer p-Smad2 cells than did those of WT mice (n = 4; P = .04). (E) Sequential dual staining with p-Smad2 and desmin antibodies showed that most of the cells expressing p-Smad2 were desmin-positive HSCs in the injured areas of the liver at 3 days after CCl4 challenge.
