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. Author manuscript; available in PMC: 2010 Sep 1.
Published in final edited form as: Cell Microbiol. 2009 Apr 30;11(9):1306–1328. doi: 10.1111/j.1462-5822.2009.01332.x

Fig. 7. Treatment of Caco-2 cell cultures with pronase, but not trypsin or PLC, enhances CPB secretion.

Fig. 7

(A) Caco-2 cells were treated with trypsin or EDTA for 15 min at 37°C. Detached cells were washed and resuspended in MEM to a final cell density of 70×105 cells/ml. Caco-2 cell cultures, MEM, trypsin-detached or EDTA-detached Caco-2 cells were then infected, in a 24-well microplate, with CN3685 for 1.5 h at 37°C. The supernatants were analyzed for CPB levels by Western blot. (B) Caco-2 cells were treated with 1×10−3 or 5×10−3 U/ml of phospholipase C (PLC) in the form of CPA for 1 h at 37°C. Cells were then washed and added with MEM. Untreated Caco-2 cell cultures, MEM or PLC-treated Caco-2 cells were infected with CN3685 for 1.5 h at 37°C. The supernatants were analyzed for CPB levels by Western blot. (C) Caco-2 cells were treated with pronase (100 µg/ml) for 20 min at 37°C. Detached cells were thoroughly washed and resuspended in MEM to a final cell density of 7×105 cells/ml. MEM, Caco-2 cell cultures or pronase-treated Caco-2 cells were then infected, in a 24-well microplate, with CN3685 for 45 min or 1.5 h at 37°C. The supernatants were analyzed for CPB levels by Western blot. Expected migration of a 35 kDa protein indicated at left of each blot.