1. Volume 39, Issue 4, Pages 528-537 (October 2003)
Regulation of ERK/JNK/p70S6K in two rat models of liver injury and fibrosis 
Gianluca Svegliati-Baroni, Francesco Ridolfi, Zaira Caradonna, Domenico Alvaro, Marco Marzioni, Stefania Saccomanno, Cinzia Candelaresi, Luciano Trozzi, Giampiero Macarri, Antonio Benedetti, Franco Folli 
Journal of Hepatology 
Volume 39, Issue 4, Pages (October 2003)
DOI: /S (03)

2. Fig. 1
ERK (A, B), p70S6K (C, D) and JNK (E, F) activity and total protein measurement after DMN treatment. (A) Phosphotransferase activity of ERK was measured in rat liver lysates using a non-radioactive assay kit (Upstate Biotechnology) according to manufacturer's instructions. (B) ERK2. Proteins (50 μg/lane) from the whole liver were separated by SDS-PAGE and immunoblotting was performed by using antibodies that specifically recognize ERK2 (right blot). (C–E) Proteins (50 μg/lane) from the whole liver were separated by SDS-PAGE and immunoblotting was performed by using antibodies that specifically recognize the phosphorylated (left blots) and non-phosphorylated (right blot) form of p70S6K (C, D) and JNK (E, F). Representative immunoblots are shown. The graph summarizes the data from three immunoblot analysis. **P<0.01 versus control; *P<0.05 versus control. In panel C, left blot, statistical analysis was performed by using the lowest detectable level as percent.
Journal of Hepatology  , DOI: ( /S (03) )

3. Fig. 2
Immunohistochemistry for phosphorylated ERK1/2 (pERK) (A–D) and p70S6K (pp70S6K) (E–H) in normal and DMN treated rats. (A–D) pERK. In control rats (A) only a faint punctate cytoplasmic pattern in hepatocytes was observed, while a strong nuclear (thin arrow) and a plasma membrane (arrowhead) positivity is evident 6 h after DMN (B). Nuclear positivity can be also detected in some non-parenchymal cells close to area of necrosis (thick arrow). Nuclear staining (arrows) is still more evident 24 h after DMN (C), while at 120 h (D) a more diffuse cytoplasmic staining (arrow) is observed. Nuclear positivity of a non-parenchymal cell close to area of necrosis is also shown (arrowhead). (E–H) pp70S6K. In normal rats pp70S6K was detected only in isolated zone 3 hepatocyte nuclei (arrow) (E), while a positive cytoplasmic and, mostly, nuclear immunostaining was evident 6 (F), 48 (G) and 120 (H) h after DMN (arrows). Final magnification, 100×.
Journal of Hepatology  , DOI: ( /S (03) )

4. Fig. 3
Immunohistochemistry for phosphorylated JNK (pJNK) (A–D). No evidence of zone 3 staining was observed in control rats (A), while nuclear localization of pJNK was detected 6 h after DMN (arrows) (B). In B, also a positive staining of a stellate-like cell can be observed (arrowhead). At 48 (C) and 120 h (D) after DMN, immunostaining for pJNK was evident in rare zone 3 hepatocytes nuclei (arrow), and in stellate-like perisinusoidal cells (arrowheads). Final magnification 100×.
Journal of Hepatology  , DOI: ( /S (03) )

5. Fig. 4
Intracellular kinases phosphorylation, αSMA and morphometry for αSMA- and PCNA-positive cells in hepatocytes (A) and HSC (B) isolated after DMN treatment. Hepatocytes (A) and HSC (B) were isolated at different time points after DMN treatment. Immunoblotting was performed on total cell lysates by using antibodies that specifically recognize pERK1/2 and ERK2, pJNK and JNK2, pp70S6K and p70S6K, and αSMA. Representative blots are shown. (C, D) Morphometry for αSMA- and PCNA-positive cells in liver sections was performed as described in Section 2. *P<0.05 versus control, **P<0.01 versus controls.
Journal of Hepatology  , DOI: ( /S (03) )

6. Fig. 5
ERK (A, B), p70S6K (C, D) and JNK (E, F) activity and total protein measurement after BDL treatment. (A) Phosphotransferase activity of ERK was measured in rat liver lysates using a non-radioactive assay kit (Upstate Biotechnology) according to manufacturer's instructions. (B) ERK2. Proteins (50 μg/lane) from the whole liver were separated by SDS-PAGE and immunoblotting was performed by using antibodies that specifically recognize ERK2 (right blot). (C–E) Proteins (50 μg/lane) from the whole liver were separated by SDS-PAGE and immunoblotting was performed by using antibodies that specifically recognize the phosphorylated (left blots) and non-phosphorylated (right blot) form of p70S6K (C, D) and JNK (E, F). Representative immunoblots are shown. The graphs summarize the data from three immunoblot analysis. **P<0.01 versus control; *P<0.05 versus control.
Journal of Hepatology  , DOI: ( /S (03) )

7. Fig. 6
Immunohistochemistry for pERK (A–D) and pp70S6K (E–H) in normal and BDL treated rats. (A–D) pERK. In control rats (A) only a weak cytoplasmic immunostaining was observed in the periportal area, while a strong nuclear positivity is evident in cholangiocytes (thick arrow) of proliferating ducts at 3 (B), 7 (C) and 28 days (D) after BDL. Rare periportal hepatocytes nuclei are also positive at day 3 (thin arrow). (E–H) pp70S6K. In sham-operated rats, no immunostaining for pp70S6K was detected (E). Three days after BDL, a specific staining appears in nuclei of proliferating bile duct cells (thick arrow) (F). At 7 and 28 days after BDL, the nuclear staining is evident both in cholangiocytes (thick arrows) and in hepatocytes (thin arrows) (G, H). Final magnification: 100×.
Journal of Hepatology  , DOI: ( /S (03) )

8. Fig. 7
Immunohistochemistry for phosphorylated JNK (pJNK) (A–D). Some mononuclear cells in the portal tract were positive (arrowhead) in sham-operated animals (I), while after BDL, pJNK was evident in the perinuclear region and in the nuclei of rare cholangiocytes (arrows), and in some inflammatory and fibroblast-like cells of the portal tract (arrowhead) at day 3 (B), 7 (C) and (28). Final magnification: 150×.
Journal of Hepatology  , DOI: ( /S (03) )

9. Fig. 8
Intracellular kinases phosphorylation, αSMA and morphometry for αSMA- and PCNA-positive cells in hepatocytes (A) and HSC (B) isolated after BDL treatment. Hepatocytes (A) and HSC (B) were isolated at different time points after DMN treatment. Immunoblotting was performed on total cell lysates by using antibodies that specifically recognize pERK1/2 and ERK2, pJNK and JNK2, pp70S6K and p70S6K, and αSMA. Representative blots are shown. (C, D) Morphometry for αSMA- and PCNA-positive cells in liver sections was performed as described in Section 2. *P<0.05 versus control, **P<0.01 versus controls.
Journal of Hepatology  , DOI: ( /S (03) )