Individual knockdown of NMD factors in MM6 cells.

<p>(A) Western blot analysis of UPF1, UPF2 and UPF3b protein expression in MM6 cells with and without knockdown of UPF1, UPF2 and UPF3b, respectively. Cell lysates (50–250 µg protein) in SDS-PAGE loading buffer (5×) were separated by 10% SDS-PAGE and transferred to a nitrocellulose membrane by electro blotting. The membrane was incubated with UPF1, UPF2, UPF3b and β-actin antibody. Visualization of protein bands was carried out by the use of infrared dye-conjugated antibodies (IRDye®), and analysis was performed with the Odyssey® infrared imaging system (<i>LI-COR</i>® Biosciences). β-actin was used as loading control. The relative changes of UPF knockdown samples to control samples (set as 1) are given as mean+SEM of three independent experiments, **p<0.01. (B) Effect of UPF knockdowns on 5-LOΔ3 and mature 5-LO mRNA expression in MM6 cells. The cells were cultured with and without TGFβ (1 ng/ml) and calcitriol (50 nM) for 24 h. Then, total RNA was isolated and cDNA was prepared by random hexamer priming. PCR analysis was carried out using primer pair G for 5-LOΔ3. For analysis of the mature 5-LO transcript, the total RNA was reverse transcribed using oligo-dT primers. PCR was performed using primer pair F and β-actin mRNA served as constitutively expressed control. Variations in the expression levels compared to non-treated samples (set as 1) are given as the mean+SE of three or four independent experiments, *p<0.05, **p<0.01.