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. 2015 Jul 14;106(8):1033–1040. doi: 10.1111/cas.12713

Figure 2.

Figure 2

CRKL is downregulated by miR-200b/200c/429. (a) The putative miR-200b/200c/429 target site in the 3′-UTR of CRKL mRNA. Seed sequences are shown in bold type. (b) CRKL protein downregulation by miR-200b/200c/429 in H1299 cells. Cells were transfected with control or miRNA mimic oligonucleotides. CRKL and β-actin levels were analyzed by immunoblotting. (c) Downregulation of CRKL mRNA by miR-200b/200c/429 in H1299 cells. Cells were transfected with control or miRNA mimic oligonucleotides for 24 h. Relative gene expression levels were quantified using the ΔΔCt method, and the results were normalized to the expression of the housekeeping gene GAPDH. The data are shown as the mean ± standard errors of three independent experiments normalized to their respective controls as 1. (d) The CRKL 3′-UTR is responsive to miR-200b/200c/429 in H1299 cells. Cells were transfected with the pMIR-REPORT vector containing the CRKL 3′-UTR (pMIR-CRKL-3′-UTR) or a mutant (pMIR-CRKL-3′-UTR-mut) along with control plasmids expressing Renilla luciferase (phRG-TK). The cells were co-transfected with control or miRNA mimic oligonucleotides. After 24 h, luciferase activity was measured using the dual-luciferase reporter assay system. All of the experiments were performed in quadruplicate, and the mean and standard deviation are indicated by the bars .