Ectopically expressed SerB binds to and dephosphorylates NF-κB p65.

<p>(<b>A</b>) 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>B</b>) Densitometry of immunoblot in A) showing ratio of immunoprecipitated NF-κB p65 relative to immunoprecipitated Myc-SerB. (<b>C</b>) 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. (<b>D</b>) 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 <i>x</i>-<i>y</i> lines indicated in C) are shown. (<b>E</b>) 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. (<b>F</b>) Densitometry of immunoblot in E) showing ratio of immunoprecipitated GFP-NF-κB p65 or p65 S536D relative to immunoprecipitated Myc-SerB.