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. 2004 Mar;15(3):1077–1088. doi: 10.1091/mbc.E03-05-0321

Figure 7.

Figure 7.

Inhibitory effect of non–trans-interacting nectin-1 on the Cef-induced formation of lamellipodia through Rac in MDCK cells. (A) The expression level of E-cadherin-GFPN. EL, wild-type MDCK, and E-cadherin-GFPN-MDCK cells were homogenized and 20 μg of each homogenate was subjected to SDS-PAGE, followed by Western blotting using the anti-E-cadherin mAb and anti-GFP pAb. (a) the anti-E-cadherin mAb; (b) the anti-GFP pAb; (1) EL cells; (2) wild-type MDCK cells; and (3) E-cadherin-GFPN-MDCK cells. The arrow and arrowhead indicate E-cadherin and E-cadherin-GFPN, respectively. (B) Effect of Cef on the formation of lamellipodia. E-cadherin-GFPN-MDCK cells were cultured on the Cef- or IgG-coated coverslips for indicated periods of time. The cells were fixed and stained with rhodamine-phalloidin. (a1) Cef at 30 min (n = 100, N:S:M = 92:8:0); (a2) Cef at 60 min (n = 100, N:S:M = 80:0:20); (a3) Cef at 90 min (n = 100, N:S:M = 35:0:65); (b1) IgG at 30 min (n = 100, N:S:M = 93:7:0); (b2) IgG at 60 min (n = 100, N:S:M = 88:12:0); and (b3) IgG at 90 min (n = 100, N:S:M = 85:15:0). (C) Effect of nectin-1, Nef-3, and N17Rac1 on the formation of lamellipodia. E-Cadherin-GFPN-MDCK cells or FLAG-nectin-1- or FLAG-N17Rac1-overexpressing E-cadherin-GFPN-MDCK cells were cultured on the coverslips coated with Cef or a mixture of Cef and Nef-3 for 90 min. The cells were then fixed and stained for F-actin and FLAG-nectin-1 or -N17Rac1 using rhodamine-phalloidin and the anti-FLAG pAb. The figures represent the rhodamine-phalloidin staining. (a) E-cadherin-GFPN-MDCK cells on the Cef-coated coverslips (n = 100, N:S:M = 44:0:56); (b) nectin-1-overexpressing E-cadherin-GFPN-MDCK cells on the Cef-coated coverslips (n = 100, N:S:M = 79:21: 0); (c) nectin-1-overexpressing E-cadherin-GFPN-MDCK cells on the coverslips coated with a mixture of Cef and Nef-3 (n = 100, N:S:M = 20:0:80); and (d) N17Rac1-overexpressing E-cadherin-GFPN-MDCK cells on the Cef-coated coverslips (n = 100, N:S:M = 95:5:0). N, no lamellipodia; S, formation of lamellipodia to a small extent; and M, formation of lamellipodia to a medium or large extent. Bars, 10 μm. The results shown are representative of three independent experiments.