PMC:3245220 / 17454-19577 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3245220","sourcedb":"PMC","sourceid":"3245220","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3245220","text":"M-CSF-Dependent Activation of PKC Does Not Regulate the Classical NF-κB p65 Activation Pathway\nCanonical activation of NF-κB occurs via phosphorylation and degradation of IκBα leading to the release and nuclear translocation of the NF-κB p50/p65 heterodimer to transactivate target genes [37], [39]. Since conventional PKC activity was important in regulating M-CSF-induced NF-κB activation, we next investigated whether IκBα degradation was regulated by PKC. Cells were treated with cyclohexamide (CHX) to inhibit protein synthesis of IκBα, and its degradation was followed. As shown in Figure 5A, PKC inhibition with Ro-31-8220 did not alter M-CSF-induced IκBα degradation, suggesting that M-CSF-induced PKC activity augmented NF-κB transcriptional activity by an alternative pathway, like post-translational modification of NF-κB p65.\n10.1371/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.\n(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.\n\nM","divisions":[{"label":"Title","span":{"begin":0,"end":94}},{"label":"Figure 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