Figure 3

Activin A activates SMAD signaling in LE6 cells. (A) LE6 cells were treated with activin A (200 ng/ml) for 6 h, 12 h, 24 h 48 h and 72 h, cell lysis was analyzed by western-blot using specific antibody to phosphorylated Rb, cyclinD1, cyclinE , p21^WAF1/Cip1, p15^INK4B p27^Kip1, CDK2 and CDK4. β-actin was used as a loading control. (B) LE6 cells were treated with activin A (200 ng/ml) for 1 h, 6 h, 12 h and 24 h, cell lysis was analyzed by western-blot using specific antibody to phosphorylated JNK1/2, ERK1/2 and p38. (C) LE6 cells were treated with activin A (200 ng/ml) for 15 min, 30 min, 60 min and 120 min, cell lysis was analyzed by western-blot using specific antibody to phosphorylated SMAD2, SMAD3. (D) LE6 cells were treated with activin A (200 ng/ml) for 60 min, cell lysis was incubated with anti-SMAD2/3 antibody and protein A/G agarose overnight, then analyzed by western-blot using specific antibody to SMAD4. (E) LE6 cells were treated with activin A (200 ng/ml) for 15 min and 30 min, Nuclear and cytosolic fractions of cells were analyzed by western-blot using specific antibody to SMAD2/3.GAPDH or laminB was used as loading control for cytosolic or nuclear fraction.