Fig. 5.

Bax is the predicted target of hsa-miR-128. a Schematic representation of Bax and its 3′UTR indicating the binding site of hsa-miR-128 as predicted. First nine nucleotides of miR-128 and its target region (Bax 3′UTR): red colored, bold shows complete complementarity. b (i) Northern blot analysis of total RNA extracted from untransfected HEK293T cells and HEK293T cells transfected with 2 or 4 μg p(128). Hybridization to the U6 small nuclear RNA is shown as a loading control. Graph shows relative hsa-miR-128 expression. *p < 0.05 versus control, (ii) Taqman assay for mature miR-128 in NCI-H460 cells showing overexpression of miR-128 in cells transfected with 2 or 4 μg p(128). c Western blot of Bax performed on total cell extracts (i) from untransfected HEK293T cells and HEK293T cells transfected with 2 or 4 μg p(128) (left panel), and (ii) NCI-H460 cells (right panel). β-actin was used a loading control. The protein band was quantified and normalized to β-actin. Graph is plotted as mean of three independent experiments. Error bars ± SEM, *p < 0.05 versus control. Normalized ID values represent normalized integrated densitometric values. d Real-time RT–PCR analysis of Bax expression in untransfected HEK293T and HEK293T cells transfected with 2 or 4 μg p(128). Data are expressed as the average ± SEM of three independent experiments performed in triplicate. *p < 0.05 versus control. e Subcellular fractionation of Bax protein in untransfected HEK293T and HEK293T cells transfected with either non-specific control or 2 or 4 μg p(128). Cytosolic and mitochondria extracts were prepared as described in “Materials and methods”. C Cytoplasmic fraction, M mitochondrial fraction. The purity of the fractions was determined by the expression of Cox 4 (mitochondrial specific protein). β-actin was used as a loading control. Blot is a typical representative of three experiment with similar results