FIGURE 4.

JMJD5 reduced level of NFATc1 protein by post-translational regulation. A, HEK293T cells were transfected with human JMJD5-specific siRNA. After 48 h of siRNA transfection, cells were also transfected with human JMJD5-specific siRNA together with FLAG-NFATc1 and FLAG-JMJD5-WT or FLAG-JMJD5-K334A. After 48 h, total cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies. β-Actin was used as a loading control for immunoblotting. An asterisk indicates a nonspecific band. B and C, total RNAs and cell lysates were prepared from two stable clones constitutively expressing JMJD5 shRNA in RAW264 cells. Expression levels of the Nfatc1 gene were detected by real time RT-PCR (left). Relative mRNA levels are represented by values that were normalized to Gapdh transcript levels. The error bars represent S.D. values from at least triplicate experiments. The p value for statistical difference was calculated using a two-tailed Student's t test. Also, the NFATc1 protein level was detected by immunoblotting with an anti-NFATc1 antibody (right). β-Actin was used as a loading control for immunoblotting. The signals obtained from the immunoblotting were quantified with the ImageJ program. Ctrl, control.