Figure 2

Time course analysis of the expression of Endo180 and marker proteins in freshly isolated and cultured rat HSCs. (A and B) Western blot analysis of expression of Endo180, desmin, GFAP, and α-SMA during culture activation of rat HSCs. At the time points shown cells were collected and cell lysates were prepared from frozen cell pellets (A) or cells were lysed directly in lysis buffer (B) and the same amount of proteins was analyzed by SDS-PAGE and Western blot using anti-Endo180 antibody. The blots were re-probed with antibodies to β-actin, used as a loading control, and marker proteins as indicated. Total non-parenchymal cells (NPC) were prepared from rat liver by collagenase perfusion as previously described [59]. (C) Densitometric analysis of Western blots showing the time-dependent increase in Endo180 protein expression in cultured rat HSCs. The ratio Endo180/β-actin bands was quantified for each day and expressed as means ± SD of seven different samples (days 1–5, and 7) or four different samples (days 0, 6 and sub-cultured). Note that there is less beta-actin in the three first lanes in Fig. 2A. However, densitometric analysis of bands showed that the amount of beta-actin at day 3 is ~ twice as much as day 7, yet no expression of Endo180 is observed at day 3. (D) RT-PCR analysis of Endo180 mRNA expression during culture activation of rat HSCs. Total RNA was extracted from cells and Endo180 and β-actin, used as an internal control, mRNAs were analyzed by RT-PCR. One representative experiment (out of three independent experiments) is shown. SC, sub-cultured (first passage).