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. 2012 Oct 12;7(10):e47312. doi: 10.1371/journal.pone.0047312

Figure 1. Smad linker phosphorylation involves GSK3 in human melanoma lines. A.

Figure 1

Inhibition of Smad2 and Smad3 linker phosphorylation in the presence of LiCl. 24 hours post seeding, WM278, WM793 and 1205LU human melanoma cell lines were serum-starved for about 16 hours, before the addition of 50 mM LiCl for 2 and 5 hours. Cells left with 50 mM NaCl for 2 or 5 hours were used as controls. Whole cell extracts were then prepared. Immunoblots were performed with antibodies against: Phosphorylated β-catenin (pβ-catenin); total β-catenin; Both phosphoSmad3 (Thr179) and phosphoSmad2 (Thr220); phosphoSmad2 (Ser245/250/255); phosphoSmad3 (Ser204); phosphoSmad3 (Ser208); Smad2 and Smad3; GAPDH. B. Inhibition of Smad2 and Smad3 linker phosphorylation in the presence of the specific GSK3 specific inhibitor, CT99021. 24 hours post seeding, WM793 and 1205LU cells were serum-starved for about 16 hours, and incubated in the absence (−) or presence (+) of 2 µM of CT99021 for two hours. Immunoblots were performed as in A. p: Phosphorylated. S: Serine; T: Threonine.