Figure 2

Differentiation or TGF-β1 treatment increases p35 expression and Cdk5 activity in MDPC-23 cells. A, Representative Western blot analysis showing activation of the TGF-β1 signaling pathway, determined by an increase of phospho-Smad2, on successive days during MDPC-23 cell differentiation. B, Upper panel shows a representative Western blot analysis of Cdk5 and p35 protein levels in undifferentiated (UD) and 5-day differentiated (D) MDPC-23 cells. Lower panel shows quantification of the Western blot for Cdk5 and p35 in control cells and differentiated MDPC-23 cells. C, Cdk5 kinase activity was measured from immunoprecipitates of control and 5-day differentiated MDPC-23 cells. D, q-PCR analysis of p35 mRNA levels normalized against S29. Total RNA was obtained from MDPC-23 cells treated with TGF-β1 (1 ng/ml) for 0, 1, 2, and 3 h. E, Western blot analysis of phospho-Smad2, Smad2, p35, Cdk5, and α-tubulin was performed in MDPC-23 cells treated with: vehicle (control), TGF-β1 (1 ng/ml), SB431542 (10 μM), and TGF-β1 (1 ng/ml) plus SB431542 (10 μM) over the course of 0, 1, 2, and 3 h. F, Quantification of Western blot of p35 and α-tubulin was performed in MDPC-23 cells treated with: vehicle (control), TGF-β1 (1 ng/ml), SB431542 (10 μM) over 24 h. G, Cdk5 kinase activity measured from immunoprecipitates of MDPC-23 cells treated with vehicle (control), SB431542 (0.1, 1, and 10 μM), TGF-β1 (1 and 10 ng/ml), and TGF-β1 (1 ng/ml) plus SB431542 (10 μM) over 24 h. All data are presented as the mean and SEM (n = 3-5). * p < 0.05, ** p < 0.01; *** p < 0.001 (t-Test); a: p < 0.01 TGF-β1 treatment vs. control; and b: p < 0.01 TGF-β1 vs. TGF-β1 plus SB431542 treatment (Bonferroni’s test after ANOVA).