Figure 3
From: NF-κB induces miR-148a to sustain TGF-β/Smad signaling activation in glioblastoma
MiR-148a directly targets SKP1 and activates TGF-β signaling. (A) Predicted miR-148a target sequence in SKP1-3′UTR and mutant containing three mutated nucleotides in the seed sequence of miR-148a (miR-148a-mut). (B) WB of SKP1 expression in negative control 1 (NC1)- or miR-148a–transduced cells or cells transfected with NC2 or miR-148a inhibitor. β-Actin was used as the loading control. (C) WB analysis of ubiquitinated Smad3 (Ub-Smad3) expression. (D) Luciferase assay of cells transfected with pGL3-SKP1-3′UTR reporter with miR-148a mimic (10 or 50 nM), miR-148a mutant, or miR-148a inhibitor (20 or 100 nM). (E) Smad luciferase reporter activity. (F) WB analysis of p-Smad2, p-Smad3, and Smad/3 expression. (G) WB analysis of Smad2/3 expression in different cellular localizations. (H) IF analysis of Smad2/3 expression. (I) GSEA plot showing that miR-148a expression correlated positively with TGF-β–activated gene signatures (PLASARI_TGFB1_TARGETS_10HR_UP) in published TCGA patient gene expression profiles (n = 538).