CMS colocalizes with the actin cytoskeleton to the pTα endocytic pathway. (A) SupT1 pre-T cells were transfected with GFP-tagged versions of either full-length CMS, a truncated CMS form encompassing the SH3ABC domains, or a C-term CMS mutant lacking the SH3 domains. Transfected cells were labeled with phalloidin-TRITC and analyzed by confocal microscopy for the coexpression of polymerized actin (F-actin) and GFP. (B) SupT1 CMS-GFP⁺ transfectants were labeled with anti-EEA1, anti-CD63, or anti-Lamp1 mAbs followed by Alexa555–anti-mouse IgGs. Coexpression of CMS-GFP with EEA1, CD63, or Lamp1 was analyzed by confocal microsopy. (C) SupT1 CMS-GFP⁺ transfectants were incubated with an anti-pTα mAb for 15 at 37°C, washed, and fixed. After fixation, internalized pTα was detected by labeling with Alexa555–anti-mouse IgGs. Colocalization to the lysosomal compartment was determined by confocal microscopy after labeling with anti-Lamp1 plus Alexa647–anti-IgG1. Data are representative of at least 3 independent experiments.