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. 2014 Oct 30;13(19):3112–3120. doi: 10.4161/15384101.2014.949527

Figure 4.

Figure 4.

L-arabinose-induced expression and secretion of ClyA in ara operon-deleted S. typhimurium. SL and SLΔara were transformed with pClyA. (A) Expression and secretion of ClyA (34 kDa) from both transformed strains (SL- pClyA and SLΔARA- pClyA) was verified by immunoblotting using an anti-ClyA antibody. Bacterial pellets (Pellet) and filtered culture medium (Filtrate) were collected at 4.5 hour after induction at 1.5 hours with or without 0.2% L-arabinose. Arrows indicate ClyA. Upper panel is Ponseau S staining. (B) Cytotoxicity due to secretion of ClyA from transformed bacteria. SL- pClyA and SLΔARA- pClyA were grown in the presence (+) or absence (-) of L-arabinose. The bacterial spent medium was filtered (0.2 μm filter), centrifuged (3,000 × g for 5 min), and concentrated to 20 mg/ml using a concentrator. The cytotoxic effects of the concentrated bacterial supernatant (0.2 mg protein) were tested using cultured CT26 cells (1 × 104). Cell death was determined using an MTT assay kit (n = 5 per group).