CD43ct is modified by SUMO-1 and localized in PML NB. (A) In vitro transcribed/translated CD43ct was sumoylated with SUMO-1 in vitro, and the products were then separated by SDS–polyacrylamide gel electrophoresis and probed with H18 antibody (lane 1) and SUMO-1 antibody (lane 2). Western blot analysis of total bone marrow cell lysates from wt (lanes 3 and 5) and CD43-deficient (lanes 4 and 6) mice is shown. Lanes 3 and 4 are whole cell lysates probed with H18 antibody. Lanes 5 and 6 are SUMO-1 immunoprecipitates probed with H18 antibody. Filled arrows indicate bands representing CD43ct modified with 1 and 3 SUMO-1. Open arrows indicate nonsumoylated CD43ct. (B) Confocal microscopy of total bone marrow cells from wt mice stained with H18 and SUMO-1 antibodies (left panel) and H18 and PML antibodies (right panel). (C) Confocal microscopy of wt (left panel) and CD43-deficient (right panel) total bone marrow cells stained with PML antibody after 24 hours in media alone. Data are representative of at least 3 repeat experiments. (D) FACS analysis of wt (blue line) and CD43-deficient (red line) total bone marrow cells stained for intracellular PML; ex vivo (left panel) and after 24 hours in media alone (right panel).