Figure 5.

Expression of α-smooth muscle actin (α-SMA) (A) and Smad2 as well as extracellular signal regulated kinase (ERK) 1 and 2 phosphorylation (B–E) in primary cultured pancreatic stellate cells (PSCs). PSCs were cultured for the indicated periods of time, and cell lysates were resolved by 8% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. After blotting, one part of the membrane (proteins > 60 kDa (B)) was incubated with a phospho-Smad2 (P-Smad2) and the other (proteins <60 kDa (C)) with a phospho-Erk 1/2 (P-ERK 1/2) specific antibody. The phospho-ERK blot was stripped and reprobed first with an anti-ERK 1/2 protein specific antibody (D) and afterwards with an antibody to α-SMA (A). Phospho-ERK levels were further analysed by scanning densitometry and related to the ERK 1 and 2 protein level (E). Therefore, the sum of the band intensities of phospho-ERK 1 and 2 (C) as well as the sum of the corresponding ERK protein band intensities (D) were determined, and the ratio phospho-ERK/ERK-protein was calculated. The results shown are representative of three independent experiments.