Fig. 4: Smurf1 and Smurf2 regulate Shh signaling through RNF220 in Daoy cells.

A–C Real-time RT-PCR (A) Western Blot (B, C) assays showing Smurf1 and Smurf2 expression in control cerebellum and Ptch1^± medulloblastoma tissues. The statistics showing relative Smurf1 and Smurf2 protein levels normalized against the corresponding α-Tubulin level was shown in (C), and the respective controls were set to 1. D–G Real-time RT-PCR (D) and Western Blot (E–G) assays showing the expression levels of RNF220 and Shh targets, including Gli1, Ptch1, and Hhip1, in Daoy cell stably transfected with shRNAs against Smurf1 or Smurf2. The statistics showing the relative protein levels of RNF220, Gli, Ptch1, and Hhip1, normalized against the corresponding α-Tubulin level was shown in (F, G), and the respective controls were set to 1. H–K Real-time RT-PCR (H) and Western Blot (I–K) assays showing the expression levels of RNF220 and Shh targets, including Gli1, Ptch1, and Hhip1, in Daoy cells transfected with expression plasmids for wild-type or E3 ubiquitin ligase-defective form of Smurf1 or Smurf2. The statistics showing the relative protein level of RNF220 (J), Gli, Ptch1, and Hhip1 (I) normalized against the corresponding α-Tubulin level. The respective controls were set to 1. L–N Real-time PCR (L) and Western blot (M, N) assays showing the expression level of Shh targets, including Gli1, Ptch1, and Hhip1, in Daoy cells stably transfected with the indicated shRNAs against RNF220, Smurf1, or Smurf2. The statistics showing the relative protein levels of Gli, Ptch1, and Hhip1 normalized against the corresponding α-Tubulin level was shown in (N). The respective controls were set to 1. β-Actin was used as a loading control for real-time RT-PCR assays. IB immunoblot, CB cerebellum, MB medulloblastoma, ns not significant. p > 0.05, **p < 0.01.