Fig. 3.

Stimulation with NMO IgG induces canonical NFκB signaling. a Lysates (50 μg per lane) from astroglial cells without stimulation (UNT), after 20-min stimulation with 20 ng/mL TNFα (TNF), as a positive control, or after stimulation with 100 μg/mL NMO IgG (NMO) for 30, 60, or 120 min were probed by immunoblot with antibodies specific for either phosphorylated IκB-α (pIκB-α) or total IκB-α. For each antibody, panels are from the same membrane and exposure. b Lysates (50 μg per lane) from unstimulated cells (UNT) or following stimulation for 5, 15, 30, 60, or 120 min with 100 μg/mL NMO IgG (NMO) or control IgG (CON) were probed by immunoblot with antibodies for either pIκB-α or IκB-α. Accumulation of pIκB-α protein was evident after stimulation with NMO IgG but not in response to the control IgG. This panel is representative of 2 independent experiments. c Cells grown on glass coverslips were probed by immunofluorescence using antibodies against the astrocyte marker glial fibrillary acidic protein (GFAP) (red) and NFκB p65 (green) after stimulation for 20 min with 20 ng/mL TNFα or stimulation for 60 min with 100 μg/mL NMO IgG (NMO) or CON IgG (CON). In some experiments, NFκB pathway inhibitors were added to the cells for 2 h prior to stimulation. Robust nuclear translocation of p65 was observed following stimulation with NMO IgG and this translocation was blocked when cells were pretreated with NFκB inhibitors. Inhibitor concentrations: 50 μM MG132 (MG), 30 μM BAY 11–7082 (BAY), 30 μM SN50, 30 μM NF-kappa-B essential modulator (NEMO) binding peptide (NBP). These panels are representative of at least 2 independent experiments performed in duplicate. DAPI-labeled nuclei are shown in blue. Scale bar: 50 μM. d Cellular homogenates were fractionated into nuclear and cytoplasmic components prior to the preparation of lysates. Fractions (10 μg per lane) from unstimulated cells (UNT), cells stimulated for 20 min with 20 ng/mL TNFα (TNF), and cells stimulated for 60 min with 100 μg/mL NMO IgG (NMO) or CON IgG (CON) were probed by immunoblot for levels of p65. The relative purity of the fractions was determined by immunoblotting for the nuclear marker NUP98 and the cytoplasmic marker α-tubulin. Increased nuclear p65 protein was observed following stimulation with NMO IgG as compared to UNT and CON IgG-stimulated samples, consistent with the immunofluorescence in c. This blot is representative of 3 independent experiments. e Lysates (50 μg per lane) were probed by immunoblot with antibodies for either pIκB-α or total IκB-α following no stimulation (UNT), after 60-min stimulation with 100 μg/mL of CON IgG or NMO IgG, or after stimulation with NMO IgG following pretreatment with NFκB pathway inhibitors. BAY, SN50, and NBP treatments reduced pIκB-α levels following NMO IgG stimulation to the levels observed in unstimulated and CON IgG-stimulated samples. MG132 treatment resulted in accumulation of pIκB-α due to proteasome inhibition, as expected. This blot is representative of at least 2 independent experiments