Figure 5  .

Regulation of MyoD expression in rat hepatic stellate cells (HSCs). (A) Immunoblot detection of MyoD protein expression in rat HSCs. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting were performed as described in methods using equal quantities of nuclear protein extracts from RAW264.7 macrophages (R), freshly isolated rat HSCs (FI), and rat HSC cultured for one (1d) or nine (9d) days on plastic. (B) Northern blot detection of MyoD mRNA in rat HSCs. Northern blotting was performed as described in methods using equal quantities of whole cell RNA extracted from freshly isolated (FI) rat HSCs and rat HSCs cultured on plastic for either seven (7d) or nine (9d) days. Filters were probed with radiolabelled cDNA probes for MyoD, Id1, and β-actin. (C) Reverse transcription-polymerase chain reaction (RT-PCR) detection of MyoD mRNA in activated rat HSCs. Following reverse transcription, equal quantities of cDNA were amplified using primers spanning nucleotides 468 to 642 of the MyoD cDNA sequence and in the presence of 0.5 to 2.5 mM MgCl₂. The single 174 bp PCR product obtained using 1.0 mM MgCl₂ was subcloned into pcDNA3 and confirmed as exon 1 derived MyoD sequence by DNA sequence analysis.