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. Author manuscript; available in PMC: 2010 Mar 1.
Published in final edited form as: J Immunol. 2009 Mar 1;182(5):2868–2878. doi: 10.4049/jimmunol.0802368

Figure 5.

Figure 5

Activation of the NF-κB signaling cascade in T1 and FM B cells. A, Sorted cells were stimulated with anti-IgM for the indicated times and whole cell lysates were probed with antibodies recognizing phosphorylation of IKKα/β, IκBα and p65. Blots were then stripped and reprobed with anti-IKKα/β, anti-IκBα and anti-p65 to assess protein loading. B, Nuclear import of NF-κB subunits. Cytoplasmic and nuclear extracts were isolated at the times indicated from sorted subsets following anti-IgM stimulation and probed with antibodies specific for c-Rel, p65 and IκBα. PLCγ2 and HDAC1 protein levels are shown as loading controls for the cytoplasmic and nuclear fractions, respectively. C, DNA-binding of NF-IκB subunits. An EMSA was performed using a radiolabeled probe containing a κB binding site and nuclear extracts from sorted WT cells incubated with or without anti-IgM for 3 h. D, Specific p65 or cRel binding was measured by supershift assay. Blots and EMSA are representative of at least 3 independent experiments.