10.1371/journal.pone.0028081.g005 Figure 5  M-CSF-induced PKC activity does not regulate IκBα degradation but regulates the phosphorylation of NF-κB p65 at Ser276.
(A) MDMs were pretreated with cycloheximide (CHX) in the absence or presence of Ro-31-8220 for 30 minutes prior to M-CSF stimulation for the indicated times. Whole cell lysates were subjected to Western blotting with anti-IκBα antibody. Data are representative of three independent experiments. (B) MDMs were pretreated with either vehicle or Ro-31-8220 for 30 minutes before the addition of M-CSF for 10 minutes. Whole cell lysates were resolved by SDS-PAGE and phospho-Ser276 or phospho-Ser536 of NF-κB p65 was detected using phospho-specific antibodies to either residue of NF-κB p65. (C) Whole cell lysates of RAW 264.7 cells treated with vehicle control or Ro-31-8220 in the absence or presence of M-CSF were subjected to Western blot analysis with phospho-Ser276 or phospho-Ser536 NF-κB p65 antibodies. (D) Cytosolic and nuclear fractions of RAW 264.7 were obtained from the treated cells and immunoblotted for phospho-NF-κB. The purity of the cytosolic and nuclear fractions was confirmed by immunoblotting with GAPDH and Lamin B, respectively. Shown are representative blots from three independent experiments.