Cell-surface CD74 initiates a signaling cascade. (A) Splenocytes from control mice were triple stained with anti-B220, anti-IgD, and anti-CD74 or isotype-matched antibodies. Dot plots show cell-surface CD74 expression on mature cells (IgD+ and B220⁺) and immature cells (IgD^– and B220⁺). (B-C) IgD⁺ B cells from control mice were stimulated for various time periods with anti-CD74 antibody (B). Control IgD⁺ B cells or B cells from CD74^–/– mice were incubated in the presence or absence of anti-CD74 antibody or irrelevant anti-ID2 antibody for 5 minutes (C). Immediately after stimulation, cells were washed and fast frozen in liquid N₂. Next, the cells were lysed as described in “Materials and methods,” and the lysates were separated on 10% (wt/vol) SDS-PAGE and blotted with anti–p-Tyr antibody followed by HRP-conjugated antimouse antibodies. The membrane was then stripped and blotted with antitubulin (C). The arrows indicate bands of 130, 72, and 55 kDa. (D-F) IgD⁺ B cells from control mice were stimulated for various time points with anti-CD74 antibody (D). CD74^–/– B cells were incubated in the presence or absence of anti-CD74 antibody (E). Control IgD⁺ B cells were incubated in the presence or absence of anti-CD74 antibody for 5 minutes in the presence or absence of the PI3K inhibitors, wortmannin (wort) or LY 294002 (LY) (F). Immediately after stimulation, cells were washed and fast frozen in liquid N₂. Next, the cells were lysed as described in “Materials and methods,” and the lysates were separated on 10% (wt/vol) SDS-PAGE and blotted with anti–p-Akt or anti-Akt antibodies. (G) Control IgD⁺ B cells or B cells from CD74^–/– mice were incubated in the presence or absence of anti-CD74 antibody or anti-CD8 antibody (isotype control) for 5 minutes. Immediately after stimulation, cells were washed and fast frozen in liquid N₂. Next, the cells were lysed and a fraction was saved for total Syk analysis. Phosphorylated proteins from the rest of the lysate were immunoprecipitated with anti–p-Tyr antibodies. Immunoprecipitates and total lysate were separated on 10% (wt/vol) SDS-PAGE and blotted with anti-Syk antibodies as described in “Materials and methods.” The intensity of the phosphorylated band following each treatment was divided by the intensity of the nonphosphorylated band in each lane. The activation-fold ratio in the absence of any treatment was normalized to 1, and the ratio for each treatment was calculated as the intensity of the treatment sample relative to 1. The results presented are representative of at least 5 different experiments.