CD74 stimulation induces its intramembrane cleavage. (A) IgD⁺ B cells from control mice were stimulated for 30 minutes with or without anti-CD74 or anti-CD8 antibodies. 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. (B-E) B cells stimulated with or without anti-CD74 antibody in the presence or absence of the Syk inhibitor, piceatannol (Pic; 10 μM) (B-C) or (D-E) in the presence or absence of the PI3K inhibitor, LY 294002 (LY; 2.5 nM). Cells were then lysed by hot-SDS and lysates were separated on Tricine gel and analyzed with IN1 rat monoclonal antibody followed by anti–rat HRP antibodies. The CD74 isoforms p31 and p41 and the released CD74 fragment (CD74-ICD) are indicated. The intensity of the CD74-ICD band following each treatment was divided by the intensity of the p31 band in each lane. The CD74/ICD 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.