Fig. 6. Knock down of Nedd4 using siRNA increases the amount of gap junction plaques at sites of appositional membranes. WB-F344 cells were either mock transfected, or transfected with non-targeting siRNA, or Nedd4 siRNA, respectively. 144 hours after transfection the expression level of Nedd4 and Cx43, as well as the cellular distribution of Cx43 was investigated. (A) Equal amounts of cell lysates were subjected to SDS-PAGE and analyzed by immunoblotting with the anti-Nedd4 antibody. Cx43 was recognized by the anti-Cx43 antibody 71-0700. As a gel-loading control the stripped blot was reprobed with an anti-actin antibody. (B) For double immunostaining in siRNA-transfected WB-F344 cells anti-Cx43 antibody C13720 (a,c,d,f) and the anti-Nedd4 antibody (b,c,e,f) were used. Cells were transfected with non-targeting siRNA (a-c) or with Nedd4 siRNA (d-f). White arrows indicate plasma membranes between adjacent Nedd4-expressing cells that show much fewer gap-junction plaques compared with plasma membranes of Nedd4-depleted cells. Bars, 20 µm.