Accumulation of <i>iNOS</i> protein following LPS requires Nocturnin.

<p>(<b>A</b>) iNOS protein is reduced in <i>Noc</i> KO MEFs following LPS treatment. WT and <i>Noc</i> KO MEFs treated with LPS for twenty-four hours were collected for protein extraction. Proteins were analyzed by Western blotting with α-iNOS antibody (a representative blot is shown in top panel) and protein levels were determined (normalized to β-tubulin levels) and plotted as mean (±SEM) of three independent MEF lines (bottom panel). (<b>B</b>) Expression of Nocturnin in <i>Noc</i> KO MEFs rescues the LPS induction of iNOS protein. <i>Noc</i> KO MEFs were electroporated with either empty FLAG vector (control) or a vector expressing a FLAG-Nocturnin fusion protein (NOC-FLAG). Forty-eight hours after electroporation, MEFs were treated for 24 hours with LPS and were then collected for protein extraction. Proteins were analyzed by Western blotting with α-iNOS and α-FLAG antibodies. Shown are extracts expressing two different levels of NOC-FLAG (compare lanes 3 and 4, top blot), which correlate with the level of rescued iNOS protein (compare lanes 3 and 4, bottom blot). The arrowhead marks a non-specific band (this band is not iNOS, as it is still present in samples from iNOS knockout mice, data not shown). The data shown are from two independently-derived WT and two independently-derived KO MEF lines (one per lane). This experiment was also repeated in a third independent MEF line for each genotype with similar results (data not shown).