Figure 3

TGF 1-dependent Smad2/3 phosphorylation is Ca ²⁺ dependent. (A) A representative Western blot demonstrating Smad 2/3 phosphorylation when cells are incubated in media either containing Ca²⁺ or without. (B). Quantification of Western blots showing that the increased phosphorylation of Smad2/3 after 60 min of TGFβ1 exposure (10 ng/ml) is largely dependent on the presence of extracellular Ca²⁺. Phosphorylation of Smad2/3 was examined by fold change over total Smad2/3 and normalized to β-actin (NFC n = 2, IPF n = 4, data pooled for statistical analysis). HLMFs stimulated for 1 h in Ca²⁺-free media phosphorylated significantly less Smad2/3 than those incubated in media containing Ca²⁺. *P < 0.05, ***P < 0.001 results are represented as median (IQR) (one-way ANOVA, corrected by Sidaks multiple comparison test).