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. 1998 Dec 8;95(25):14979–14984. doi: 10.1073/pnas.95.25.14979

Figure 1.

Figure 1

Cleavage of E-cadherin, but not occludin, by BFT. (A) Occludin immunoblot of HT29/C1 cells. HT29/C1 cells were treated with BFT (5 nM) for various times, lysed, and examined by Western blotting using a polyclonal anti-human occludin antibody. Occludin is a 65-kDa protein (arrow). Lane 1, untreated HT29/C1 cells; lanes 2–5, BFT for 30 min, 60 min, 2 hr, 3 hr, respectively; lane 6, untreated HT29/C1 cells. (B) Time course of BFT proteolysis of E-cadherin. HT29/C1 cells were treated for various times with BFT (5 nM), lysed in 1× SDS/gel loading buffer, and examined by Western blot using the E2 antibody (34). Cell-shape changes were observed beginning 10 min after treatment with BFT, with 100% of cells affected by 30 min. Arrows indicate intact E-cadherin (120 kDa) and E-cadherin fragments (33 and 28 kDa) observed after BFT treatment. Lane 1, untreated HT29/C1 cells; lanes 2–10, BFT for 1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 1 hr, 2 h, 3 hr, respectively. Immunostaining of the housekeeping protein GAPDH showed no change over time. (C) Concentration dependency of proteolysis of E-cadherin by BFT. HT29/C1 cells were treated with 0.005–5 nM BFT for 30 min. The immunoblot was processed and probed with E2 antibody as in 1B. Lane 1, untreated HT29/C1 cells; lane 2, 0.005 nM BFT; lane 3, 0.05 nM BFT; lane 4, 0.5 nM BFT; lane 5, 5 nM BFT. (D) Time course of BFT proteolysis of E-cadherin expressed by LE cells. LE cells (E-cadherin-transfected L cells) were induced overnight with 1 μM dexamethasone followed by treatment for various times with BFT (5 nM). The immunoblot was processed and probed with the E2 antibody as in Fig. 1B. Lane 1, control LE cells; lanes 2–6, BFT for 5 min, 10 min, 15 min, 30 min, 1 hr, respectively. (E) Effect of CCCP on BFT-stimulated E-cadherin proteolysis. HT29/C1 cells were treated with CCCP as described in Materials and Methods. CCCP-treated HT29/C1 cells were compared with HT29/C1 cells treated with BFT (5 nM) in standard HT29/C1 medium. Lanes 1–4, HT29/C1 cells without CCCP treatment. Lane 1, control HT29/C1 cells; lanes 2–4, BFT for 10 min, 30 min, 60 min, respectively. Lanes 5–8, HT29/C1 cells treated with CCCP. Lane 5, BFT for 10 min, 30 min, 60 min, control HT29/C1 cells, respectively.