Fig. 6

CrmD N77F mutant blocks chemokines but lacks anti-TNF activity. a Coomasie blue stained gel showing 500 ng of recombinant CrmD wild type (WT) and N77F mutant expressed by recombinant baculoviruses. Molecular mass standards are shown in kDa. b CrmD N77F binds mouse Ccl25 but not mouse TNF (mTNF). The binding of 100 nM Ccl25 and mTNF to CrmD WT or N77F mutant was assessed by SPR experiments. c CrmD N77F mutant does not interfere with mouse TNF-mediated cytotoxicity on L929 cells. The cell viability of L929 cells after a 18 h incubation with 1.2 nM TNF in the absence or presence of the indicated molar ratios of CrmD variants was determined. Data is represented as mean ± standard deviation (SD) of the % relative to cells incubated without TNF (media). d CrmD N77F blocks mouse Ccl25-induced migration of MOLT-4 cells. The number of cells that migrated through the transwell filter after 4 h incubation at 37 ^oC with 50 nM mouse Ccl25 in the absence or presence of the indicated increasing molar ratios of CrmD variants is shown. Data is represented as mean ± SD of triplicates. In c and d, one experiment representative of three independent experiments is shown