Figure 5.

N-cadherin knockdown restores gap junction assembly in RL-NΔE cells. Cells were infected with ShEGFP and ShN-Cad and selected as described in Materials and Methods. Expression level of total (T), detergent-soluble (S), and detergent-insoluble (I) N-Cad (A) and Cx43 (B) were determined in cells infected with ShEGFP and ShN-Cad, respectively. Ten micrograms of total protein and normalized soluble and insoluble fractions were resolved on 12% SDS-PAGE and immunoblotted for Cx43 and N-Cad. The blots were reprobed with β-actin to verify equal loading. Note that the N-Cad expression level is significantly reduced in ShN-Cad infected pooled RL-NΔE polyclonal cultures (A), whereas the expression level of Cx43 (B) is not discernibly affected. Note also that knock down of N-Cad decreased the detergent soluble fraction, and increased the detergent-insoluble fraction, of Cx43. (C) Pooled polyclonal cultures of RL-NΔE cells infected with ShEGFP and ShN-Cad were immunostained for Cx43 and N-Cad. Note the reduced expression of N-Cad at areas of cell–cell contact and in the intracellular pool, the disappearance of intracellular Cx43, and GJ formation in cells infected with ShN-Cad but not with control retrovirus (ShEGFP). Bar = 15 μm.