CDK6 contributes to basal and TNF-inducible p65 Ser536 phosphorylation.

<p>(<b>A</b>) MDCK cells were treated for 24 h with siRNAs directed against CDK6 or adequate scrambled controls followed by treatment with TNFα as indicated. Total cell extracts were analyzed for phosphorylation of p65 and expression of the indicated proteins by immunoblotting. Numbers indicate relative levels of p65 Ser536 phosphorylation. (<b>B</b>) The experiment was done as in (A) with the exception that cells were fractionated in nuclear and cytosolic extracts. Extracts were further analyzed for the occurrence of nuclear p65, relative levels are indicated by numbers. Suppression of CDK6 in the cytoplasm was validated by CDK6 antibodies and quantified. Actin and HDAC1 detection served to control the purity of the fractions. (<b>C</b>) A Hela cell line stably transfected with shRNAs directed against CDK6 (shCDK6, K06) or cells stably transfected with pSuper-Puro (vector) were subjected to cell cycle arrest by serum deprivation for 48 h (arrest). Thereafter cells were released for 6 h by addition of 20% serum (G1 release). In addition, cells were treated with IL-1 (10 ng/ml) for 30 min as indicated. Total cell extracts were separated by SDS-PAGE in two sets on one gel and were transferred to one membrane. One half of the membrane was probed with antibodies against phospho-Ser536, CDK6, CDK4 and β-actin. The other half was probed with anti p65 antibodies. Numbers indicate relative phosphorylation of p65 or relative protein levels of p65 and CDK6. The graph shows the mean +/− s.e.m. of relative Ser536 phosphorylation normalized to p65 total protein as determined in three independent experiments. The asterisk indicates significant differences (p< = 0.018) as determined by paired t-test. (<b>D</b>) Naturally KSHV-infected primary effusion lymphoma (PEL) cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051847#pone.0051847-Arvanitakis1" target="_blank">[39]</a> were stably infected with lentiviruses encoding shRNAs directed against CDK6 or scrambled shRNA controls. Cells were fractionated into cytosolic (c), nuclear soluble (ns) and nuclear insoluble (ni) fractions. These extracts were analyzed by immunoblotting with the indicated antibodies. Tubulin was used as a loading control for cytosolic and nuclear soluble fractions and SP1 as a marker for the nuclear insoluble fraction. Numbers indicate relative levels of phospho-Ser536 p65, cyclinD3 and CDK6. (<b>E</b>) PEL cells silenced for CDK6 expression as described in (D) were analyzed by indirect immunofluorescence for p65 and p65 Ser536 phosphorylation with specific antibodies, nuclear DNA was revealed by DAPI staining.