Alternative CD5 E1B motif encodes a truncated isoform of CD5 protein starting at exon 3 and retained intracellularly. (A) In vitro translation of CD5 using pLuc (C+), pDNRdual (C–), CD5-1B pDNRdual (CD5-1B) using the TNT T7-coupled reticulocyte system. (B) Precipitation of CD5 in T cells and 697 pre-B cells using antiphosphotyrosine (PY). MW indicates molecular weight; IP, immunoprecipitation. (C) FACS analysis of Jurkat T and 697 pre-B cells for CD5 protein expression in the absence (membrane CD5) or in the presence (intracellular CD5) of saponin. Filled histograms denote CD5 staining; open histograms, isotypic controls. (D) Immunohistochemical staining with UCHT2 of saponin-treated 697 pre-B cells. Images were acquired as in Figure 3B. (E) Western blot analysis of COS-1 cells transfected with EGFP-expressing vector, or fusion CD5 E1B transcript and EGFP. Top arrow indicates CD5-1B–EGFP; bottom arrow, EGFP. (F) Confocal microscopy analysis (× 1000) of the subcellular localization of fusion CD5 E1B transcript and EGFP in COS-1 cells. Confocal images were acquired using constant settings on a Leica TCS-NT fluorescence microscope with a 100 ×/1.40 NA oil objective using a FITC filter and Leica TCS-NT software version 1.6.587 (Leica Microsystems, Bensheim, Germany). Image sizes were adjusted using Adobe Photoshop.