Figure 2

Assessment of NF-κB activation in human B lymphocytes by multiparameter intracellular flow cytometric analysis of IκB isoforms (-α, -β, -ε) and the phosphorylation status of IκBα. R2G6 cells (3 × 10⁵) were incubated with rCD154 in the presence or absence of 30 μM of the proteosome inhibitor lactacystein (LAC) or the inhibitor of IκB phosphorylation BAY11-7082 (BAY; Calbiochem) for 15 min at 37°C. Cells were fixed, permeabilized and stained with isotype-matched control antibody or antibody that recognize (a) IκBα, pIκBα, (b) IκBβ or IκBε. Two independent experiments are presented in both (a) and (b). Experiment 1 in (a) depicts IκBα staining following CD154 stimulation in the presence or absence of LAC. Experiment 2 in (b) depicts IκBα and pIκBα staining in the presence or absence of LAC. It is important to note that R2G6 cells are known to have a high level of constitutive NF-κB activation, accounting for the large percentage of cells expressing p-IκBα. Experiment 1 in (b) depicts staining for IκBβ following stimulation with CD154 in the presence or absence of LAC. Experiment 2 in (b) depicts staining for IκBε following stimulation with CD154 in the presence or absence of LAC. Dot plots of cellular size or complexity on the x-axis versus fluorescence intensity in the channel that detects the fluorochrome conjugated secondary antibody on the y-axis are shown in (a) and (b). Insets of histograms of fluorescence intensity of the channel that detects the fluorochrome conjugated secondary antibody are shown in (a) and (b). The percentage of positive cells and the mean fluorescence intensity (MFI) of positive staining are indicated. The solid line indicates the division between negative background staining of isotype-matched control antibody and positive staining with the antibody that recognizes a specific IκB. (c) Western blot analysis of pIκBα as well as total IκBα, -β and -ε following CD154 stimulation in the presence or absence of LAC or BAY11-7082 (BAY).