Increase of PLD4 level by LPS stimulation in MG6 cells.

<p>MG6 cells were treated with LPS (500 ng/ml) or vehicle (PBS) for 24 hrs. (A) The Western blot analysis (15 µg proteins, 10.5% SDS-PAGE) using anti-PLD4 antibody revealed that the levels of PLD4-related bands (70–80 kDa) were increased in LPS-stimulated MG6 cells (left) compared with those in control cells (right). (B) Whole cell homogenates (W), nuclear fractions (N), and supernatants (C) were prepared. Mouse spleen homogenate (S) was prepared as a positive control. Each sample was deglycosylated by peptide-N-glycosidase F (PNGase F). Western blot analysis (15 µg proteins, 10.5% SDS-PAGE) using PLD4 antibody revealed that PLD4-positive bands were found in the nuclear fraction and supernatants. After PNGase F treatment, PLD4-positive band sizes were changed (48 kDa). (C) Intensity of each band was measured and relative increase of PLD4 by LPS-stimulation in each fraction was expressed graphically. The data are presented as mean ± SE of four experiments. Asterisk and double asterisk in C indicate P<0.01 and P<0.05 by Mann-Whitney's U test, respectively. Levels of PLD4 were increased in all LPS-stimulated MG6 cell samples compared with those in control cell samples.