Fig. 3.

BMP7 activates JNK signaling in SIX2⁺ NZ cells. (A) Western blot analysis showing expression of TAB1, TAK1 and XIAP in NZ cells. HEK293 cell lysate was used as a positive control (Blonska et al., 2005; Dan et al., 2004; Ge et al., 2003). β-tubulin shows protein loading. (B) Western blot analyses of activated JNK and p38 in NZ cells stimulated with 50 ng/ml BMP7 for 5-30 minutes. β-tubulin was used as loading control. (C) Western blot analysis of phosphorylated SMAD1/5/8 in NZ cells stimulated with 50 ng/ml BMP7 for 5-45 minutes. β-tubulin shows protein loading. (D) Western blot analyses of phosphorylated forms of Jun and ATF2 in NZ cells stimulated with 50 ng/ml BMP7 for 5-30 minutes. β-tubulin was used as loading control. (E) Western blot analyses of NZ cells transduced with control GFP- or dominant-negative (dn) TAK1 adenoviral (Ad) vectors. Cells were stimulated with 50 ng/ml BMP7 for 5-30 minutes and subsequently analyzed for phosphorylated JNK. Lane C, unstimulated control. HA-tagged dnTAK1 was detected using anti-HA antibody. β-tubulin was used as loading control. (F) IF staining of unstimulated NZ cells (i) or stimulated with 50 ng/ml BMP7 for 10 minutes (ii). Phosphorylated JNK (red), SIX2 (green), DAPI (blue). Arrows indicate SIX2⁺ cells, which robustly activate the JNK pathway upon BMP7 stimulation. Scale bars: 50 μm.