Fig. 5

Pinch loss impairs TGF-β1/Smad2/3 signaling. a Immunofluorescence (IF) staining. ATDC5 cells (2 × 10⁴ cells/well) were seeded in confocal dishes (SPL Life Science) for 24 h, and then subjected to IF staining using the indicated antibodies. Scale bar, 20 µm. b Immunoprecipitation (IP) assay. COS-7 cells (2 × 10⁶ cells/well) were seeded in 100-mm dishes. Twenty-four hours later, the cells were transfected with 5 μg Pinch1 expression plasmids. After 24 h, whole-cell extracts were prepared, immunoprecipitated with a Smad2/3 antibody, and subjected to western blot analysis using a Pinch1 (top) or Smad2/3 (bottom) antibody. c IP assay. Whole-cell extracts from ATDC5 chondrocyte-like cells were immunoprecipitated with a Smad2/3 antibody and then subjected to western blot analysis using a Pinch1 (top) or Smad2/3 (bottom) antibody. d IP assay. COS-7 cells were transfected with the indicated Flag-Pinch1 deletion mutant expression vector. After 36 h, whole-cell protein extracts were immunoprecipitated with an M2 antibody and then subjected to western blot analysis using an M2 (top) or Smad2/3 antibody (bottom). e, f Cycloheximide (CHX) experiment. Primary Pinch2 KO BMSCs were transfected with control or Pinch1 siRNA. Twenty-four hours later, the cells were treated with 10 μg·mL⁻¹ CHX. Cell lysates were subjected to western blot analysis of Smad2/3 expression. Quantitative analysis of Smad2/3 expression, normalized to β‐actin, from three independent experiments (f). g, h Smad2/3 ubiquitination. Primary BMSCs from Pinch2 KO mice were transfected with control (si-NC) or Pinch1 siRNA (si-Pinch1). Twenty-four hours later, whole‐cell extracts were immunoprecipitated with an anti‐Smad2/3 antibody, and then subjected to western blot analysis of ubiquitin (Ub) (g). Quantitative analysis of (Ub)n‐Smad2/3 from three independent experiments (h). *P < 0.01 versus si-NC. i, j IF staining. Primary BMSCs from Pinch2 KO mice were transfected with si-NC or si-Pinch1. Twenty-four hours later, the cells were treated with or without 10 ng·mL⁻¹ TGF-β1 for 30 min and then subjected to IF staining using a Smad2/3 antibody and phalloidin. Scale bar, 20 µm. Quantitative analysis of pSmad2/3 expression normalized to tubulin, from three independent experiments (j). k, l Western blotting. Primary BMSCs from Pinch2 KO mice were transfected with si-NC or si-Pinch1. Twenty-four hours later, the cells were treated with 10 ng·mL⁻¹ TGF-β1 for 30 min and then subjected to western blotting. Quantitative analysis of pSmad2/3 expression normalized to tubulin, from three independent experiments (l). *P < 0.01 versus si-NC