The CD74 signaling cascade induces the intramembrane cleavage. (A) Purified IgD⁺ B cells were preincubated with or without chloroquine (100 μM) for 1 hour. Cells were then lysed by hot-SDS, and lysates were separated on Tricine gel and analyzed with the IN1 rat monoclonal antibody, which recognizes the CD74 cytosolic domain, followed by anti–rat HRP antibodies. Positions of the CD74 isoforms, p31 and p41, and of the released CD74 fragment (CD74-ICD) are indicated. (B-C) IgD⁺ B cells from control mice and CD74^–/– B cells were pretreated with or without chloroquine. Cells were then incubated with or without anti-CD74 antibody for 5 minutes. Immediately after stimulation, cells were washed and fast frozen in liquid N₂. (B) 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. (C) Next, the cells were lysed, and lysates were separated on 10% (wt/vol) SDS-PAGE and blotted with anti–p-Akt or anti-Akt antibodies. 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 3 different experiments.