Involvement of TRAF2/5 in LMP1-mediated suppression of SAP on H9 cells. (A) The association between TRAF2/5 and LMP1 on H9 cells. H9 cells were transfected with either pSG5 or pSG5-LMP1 constructs. Cell lysates were immunoprecipitated with antibodies against TRAF2 or TRAF5. The immunoprecipitates were examined by immunoblot with LMP1 antibody. (B) Double immunofluorescence staining revealed colocalization of TRAF2/5 and LMP1 on H9 T cells. Cells were immunostained by antibodies of TRAF2/5 and LMP1, and were observed under confocal microscopy. In LMP1-H9 cells, aggregation and colocalization of TRAF5 or TRAF2 with LMP1 proteins near the cell membrane are shown as indicated by arrows. Original magnification: × 1000 (objective lens: 200 ×). Cells were imaged under Bio-Rad MRC-100 confocal laser scanning (Bio-Rad Microscience Division, Cambridge, MA). The system was connected to a Nikon Diaphot 200 inverted microscope (Nikon, Melville, NY). Images were collected using COMOS software (Bio-Rad) and assembled for publication using Adobe Photoshop 6.0 software (Adobe Systems, San Jose, CA). (C) Recovery of SAP expression on LMP1-H9 cells by TRAF2/5 dominant (DN) mutants. H9 cells were transfected with pSG5 and pSG5-LMP1 along with TRAF2/5 dominant-negative mutant constructs. The expressions of SAP protein were reduced by LMP1 and this inhibitory effect could be rescued by cotransfection with TRAF2/5 dominant-negative mutants. Error bars representing the standard deviation of 3 independent experiments are shown.