The Serine Phosphatase SerB of Porphyromonas gingivalis Suppresses IL-8 Production by Dephosphorylation of NF-κB RelA/p65
Figure 2
Ectopically expressed SerB binds to and dephosphorylates NF-κB p65.
(A) TIGKs transfected with empty vector or Myc-SerB were stimulated with TNF-α (10 ng/ml) for 5 min or left unstimulated. Cells were cross-linked with DSP and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoblots (IB) show cell lysates prior to immunoprecipitation (5% input) and immunoprecipitate (IP) with Myc antibodies. Result is representative of 3 biological replicates. (B) Densitometry of immunoblot in A) showing ratio of immunoprecipitated NF-κB p65 relative to immunoprecipitated Myc-SerB. (C) Confocal microscopic images of TIGKs expressing GFP-NF-κB p65 (green) and either Myc (Vector) or Myc-SerB. At 36 h after transfection, cells were stimulated with TNF-α (10 ng/ml) for 5 min or left unstimulated. Cells were fixed and stained with anti-Myc (red). Bars = 5 µm. Data shown are representative of 2 biological replicates. (D) The intensity (Olympus FluoView software) of the fluorescence signals of GFP-NF-κB p65 (green) and either of Myc (red, Vector) or Myc-SerB (red) on the x-y lines indicated in C) are shown. (E) TIGKs transfected with empty vector or Myc-SerB, and GFP-NF-κB p65 or GFP-NF-κB p65 S536D, were cross-linked with DSP and cell lysates were immunoprecipitated with anti-Myc antibody. Immunoblots (IB) show cell lysates prior to immunoprecipitation (5% input) and immunoprecipitate (IP) with Myc antibodies. Result is representative of 2 biological replicates. (F) Densitometry of immunoblot in E) showing ratio of immunoprecipitated GFP-NF-κB p65 or p65 S536D relative to immunoprecipitated Myc-SerB.