Repression of Apg-2 and ZO-1 by regulated RNA interference. Stable MDCK cell lines expressing the tetracycline repressor as well as shRNAs targeting ZO-1 or Apg-2 (z2 and z5 target different sequences), or a non-targeting shRNA under the control of a tetracycline-regulated promoter were plated in low calcium medium onto permeable culture inserts in the presence or absence of tetracycline. After 18 hours, the cells were switched to normal calcium medium for 24 hours adding tetracycline as indicated. The cells were then lysed in SDS-PAGE sample buffer and analysed by immunoblotting. Shown are immunoblots for Apg-2, ZO-1, ZO-2, ZO-3 and α-tubulin. Expression of ZO-1 was reduced to 24 +/- 7% and of Apg-2 to 22 +/- 6% of the respective levels in control cultures (n = 4).
Aijaz et al. BMC Cell Biology 2007 8:49 doi:10.1186/1471-2121-8-49