PECAM-1 is a potent inhibitor of cytochrome c release without blocking translocation of Bax to the mitochondrial membrane. (A) The 293 cells were transfected with PECAM-1, GFP-Bax, or both and analyzed by immunofluorescence microscopy following addition of Texas red—conjugated goat-antimouse IgG to detect cell-surface PECAM-1 (cells were not permeabilized). Expression of PECAM-1 (B) failed to significantly affect the mitochondrial staining pattern of Bax (C), and little or no colocalization was evident in the merged image (D). Original magnification, × 100. (E) Coprecipitation analysis of Bax and PECAM-1. PECAM-1 immunoprecipitates did not contain detectable levels of Bax, and Bax immunoprecipitates were devoid of PECAM-1. (F) Cells transfected as indicated were fractionated into cytosolic and heavy mitochondrial membrane fractions and each fraction analyzed for the presence of Bax and cytochrome c. Overexpression of Bax resulted in significant accumulation in the mitochondria-enriched membrane fraction and concomitant release of cytochrome c into the cytosolic fraction. PECAM-1 suppressed cytochrome c release but failed to prevent translocation of Bax into mitochondria. ITIM-less PECAM-1_{Tyr663, 686Phe}, which is not cytoprotective (Figures 4, 5, 6), failed to block either Bax translocation or cytochrome c release, as expected.