Fig. 4

MiR-4732-3p directly regulates SMAD2 and SMAD4. a Alignment of miR-4732-3p and predicted target sites in the 3’ UTR of SMAD2 and SMAD4, with mutated binding sites underlined. b Relative changes of reporter activities in K562s co-transfected with luciferase constructs and indicated microRNA mimics. A portion the SMAD2 and the SMAD4 3’UTRs were cloned downstream of Renilla in separate psiCheck-2 vectors (n = 4) (*p < 0.05, **p < 0.01). c Relative changes of reporter activities in K562s co-transfected with luciferase constructs and microRNA-blocking antisense oligonucleotides (ASOs) (n = 4) (***p < 0.001). d Relative changes of reporter activities in K562s transfected with wild-type (WT) or mutated (MUT) SMAD2 or SMAD4 3’UTR constructs (n = 4) (*p < 0.05). e Western blots of SMAD2 and SMAD4 protein expression 24 h after transfection of K562s with indicated microRNA mimics or f microRNA ASOs. α-tubulin was used as a loading control for normalization. Protein desitometric values are listed. The fold change of expression for each treatment group was first normalized to alpha tubulin input for that treatment, then ratio set relative to the control treatment