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J. effect on caspase activation. Furthermore, we found that SubAB induced focal adhesion kinase fragmentation, which was mediated by a proteasome-dependent pathway, and caspase activation was suppressed in the presence of proteasome inhibitor. Thus, 1 ITG serves as a SubAB-binding protein and may interact with SubAB-signaling pathways, leading to cell death. Our results raise the possibility that although BiP cleavage is necessary for SubAB-induced apoptotic cell death, signaling pathways associated with functional SubAB receptors may be required for activation of SubAB-dependent apoptotic pathways. Subtilase cytotoxin (SubAB) was first identified as a product of Shiga-toxigenic (STEC) O113:H21, which caused an outbreak of hemolytic-uremic syndrome (HUS) (58). Subsequently, SubAB was found only in STEC strains. Recently, however, SubAB was identified in Shiga toxin (Stx)-negative strains isolated from unrelated cases of childhood diarrhea (70). SubAB cleaved the molecular chaperone BiP, which triggered an endoplasmic reticulum (ER) stress response (57, 73). It also caused other effects, including transient inhibition of protein synthesis (51), G0/G1 cell cycle arrest (50, 51), caspase-dependent apoptosis via mitochondrial membrane damage (45), activation of the Akt-NF-B signaling (78), and downregulation of gap junction expression (32). In addition, high concentrations of SubAB induced vacuole formation in Vero cells (51, 76). Although several studies have examined the molecular mechanisms responsible for ER NSC697923 stress-induced cell death (61, 67, 74), the relationship between perturbation in protein folding in the ER following SubAB-induced BiP cleavage and activation of death pathways remains poorly understood. We found, however, that SubAB-induced apoptosis in Vero cells was caused by cytochrome release via mitochondrial permeabilization, followed by caspase activation (45). It is well-known that cell surface receptors are responsible for bacterial toxin NSC697923 binding and entry into cells, effects on various signal transduction pathways, and morphological changes of the target cell. SubB has a strong preference for binding to cell surface glycans terminating in the sialic acid release, and caspase activation. MATERIALS AND METHODS Subtilase cytotoxin preparation. producing recombinant His-tagged wild-type SubAB and catalytic inactivated mutant SubA(S272A)B (mSubAB) were used as the source of toxin for purification, according to a published procedure (51). Antibodies and other reagents. Anti-NG2 chondroitin sulfate proteoglycan antibody (AB5320), which recognizes both intact proteoglycan and core protein, was purchased from Millipore; anti-cleaved caspase-7, anti-cleaved procyclic acidic repetitive protein (PARP), anti-Bax, anti-Bak, anti-focal adhesion kinase (anti-FAK), and anti-Met antibodies were from Cell Signaling; mouse monoclonal antibodies (MAbs) reactive with NG2 (LHM2), 1 integrin (P5D2), 2 integrin (C-9), and cytochrome (7H8) were from Santa Cruz Biotechnologies; rabbit polyclonal antibodies reactive with GAPDH (FL335), normal mouse IgG, and normal rabbit IgG were from Santa Cruz Biotechnologies; mouse monoclonal antibodies NSC697923 reactive with BiP/GRP78 and conformation-specific anti-active Bax (clone 3) were from BD Biosciences. NSC697923 Conformation-specific anti-active Bak (Ab-2) antibody was purchased from Calbiochem; anti-L1CAM monoclonal antibody was from eBioscience. Caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (methoxy) fluoromethylketone (Z-VAD-FMK, or ZVAD) was purchased from BD Biosciences. Calpain inhibitor 1 (agglutinin-agarose column (bed volume, 2 ml; Seikagaku Corporation). The column was washed with 10 ml of Sol buffer, and then Sol buffer containing 1% chitooligosaccharide was used to elute the carbohydrate-containing proteins in 1-ml fractions. To confirm the presence of p250 in eluted fractions, proteins in the effluents were immunoprecipitated with SubAB as described previously (76). After SDS-PAGE, proteins were Rabbit Polyclonal to CRMP-2 (phospho-Ser522) transferred to PVDF membranes, which were incubated with streptavidin-HRP. Biotinylated p250 was detected using enhanced ECL. To identify p250, proteins in effluents were precipitated with chloroform-methanol (72). The precipitated samples were heated in SDS-PAGE sample buffer, separated in gels,.