Figure 1
Assessment of extracellular regulated kinase (ERK), jun N-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK) activity in human B lymphocytes by intracellular flow cytometric analysis. R2G6 cells (3 × 105) were fixed, permeabilized, incubated in the presence or absence of a blocking peptide and stained with isotype matched control antibodies or phosphospecific antibodies that recognize pERK, pJNK and p-p38 (a) immediately or (b) following incubation with increasing amounts of rCD154 for 15 min at 37°C. Dot plots of cell size 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). 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 pMAPK antibody. (c) Western blot analysis of pERK, pJNK or p-p38 expression in R2G6 cells following incubation with rCD154 for 15 min at 37°C.