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. 2012 Oct 26;20(3):430–442. doi: 10.1038/cdd.2012.135

Figure 2.

Figure 2

miR-125b downregulates Mad1 in a specific manner. (a) MAD1 mRNA level is reduced in presence of ectopic miR-125b. UPCI:SCC084 cells were transiently transfected with 0, 0.25, 0.5, 1 and 2 μg of miR-125b expression plasmid. Total RNA isolated was reverse transcribed and the cDNA subjected to RT-PCR using MAD1-specific primers. Relative expression values were normalized to those of GAPDH. (b) Representative picture showing that Mad1 protein level decreases upon ectopic expression of miR-125b. UPCI:SCC084 cells were transiently transfected with 0, 0.25, 0.5, 1 and 2 μg of miR-125b expression plasmid. Cell lysates were prepared followed by western blot with antibodies against Mad1 and β-actin. (c) BUB3 mRNA level is unaffected by ectopic miR-125b. UPCI:SCC084 cells were transiently transfected with 0, 0.25, 0.5, 1 and 2 μg of miR-125b expression plasmid. Total RNA isolated was reverse transcribed and the cDNA subjected to RT-PCR using BUB3-specific primers. Relative expression values were normalized to those of GAPDH. (d) Representative picture showing that Bub3 protein level is unaffected upon ectopic expression of miR-125b. UPCI:SCC084 cells were transiently transfected with 0, 0.25, 0.5, 1 and 2 μg of miR-125b. Cell lysates were prepared followed by western blot with antibodies against Bub3 and β-actin. (e) MAD1 mRNA level remains unaltered in presence of an unrelated miRNA, miR-133b. UPCI:SCC084 cells were transiently transfected with 0, 0.25 and 0.5 μg of pSB-miR-133b. Total RNA isolated was reverse transcribed and the cDNA subjected to RT-PCR using MAD1-specific primers. Relative expression values were normalized to those of GAPDH. (f) Representative picture showing that Mad1 protein level is unaffected upon ectopic expression of pSB-miR-133b. UPCI:SCC084 cells were transiently transfected with 0, 0.25 and 0.5 μg of pSB-miR-133b. Cell lysates were prepared followed by western blot with antibodies against Mad1 and β-actin. For a, c and e, data represent three independent experiments (N=3) and are shown as average±S.E. For b, d and f, images are representative of three independent experiments