Less endosomal Numb and more ubiquitylated membrane pre-TCR in fetal dnNb TG thymocytes. (A) Confocal images of fetal thymic frozen sections stained with antibodies against Numb (red), c-Cbl (green), and CD25 (blue). (B) Average number of CD25⁺ cells containing from zero to 4 c-Cbl⁺, Numb⁺, and c-Cbl⁺Numb⁺ vesicles. Four independent images for each line (WT and dnNb TG) were processed. For colocalization, analysis masks for c-Cbl (green) and for Numb (red) were made with Photoshop 7.0. Numbers of vesicles per CD25⁺ cell in each mask were counted. The same analysis was performed in masks containing Numb and c-Cbl together (yellow). An average of 34 and 45 CD25⁺ cells per image were counted for WT (black bars) and dnNb TG (gray bars), respectively. (C) Confocal images of fetal thymic frozen sections of WT (left) and dnNb TG (right) mice, stained with antibodies against ubiquitin (red), pre-TCR (green), and CD25 (blue). White arrows point to cells with intracellular ubiquitin⁺/pre-TCR⁺ vesicles (left) or membrane ubiquitin⁺/pre-TCR⁺ accumulations (right). All data are representative of at least 3 independent experiments.