Fig. 4. Loss-of-gap-junction-function Cx45.6 mutants stimulate the expression of MIP(AQP0) to levels comparable to those of wild-type Cx45.6. Lens primary cultures were infected with recombinant retroviruses RCAS(A), RCAS(A)-Cx45.6, RCAS(A)-Cx45.6(D47A) or RCAS(A)-Cx45.6(P88S) for 8 days. (A) Crude membrane preparation from cells infected with retroviruses RCAS(A)-Cx45.6(D47A) (lane 1), RCAS(A)-Cx45.6(P88S) (lane 2), RCAS(A)-Cx45.6-WT (lane 3) and RCAS(A) vehicle (lane 4) were loaded on SDS-PAGE gels and immunoblotted with antibodies against Cx45.6, -actin (A) or MIP(AQP0) (B). The Cx45.6 (A, right panel) and MIP(AQP0) (B, lower panel) bands from three separate western blot analyses were quantified by densitometry. The data are presented as the mean±s.e.m.; n=3. ^*P<0.05, in comparison with the non-Cx45.6-overexpressing control. (C) Primary cells were immunolabeled with monoclonal antibody against MIP(AQP0). The primary antibody was detected by fluorescein-conjugated anti-mouse IgG. The phase-contrast (upper panel) and immunostaining (lower panel) images were captured by a fluorescence microscope. The size of the MIP(AQP0)-stained area was quantified (UTHSCSA ImageTool Software) and presented as a percentage in the x-axis (C, right panel). The data are presented as the mean±s.e.m.; n=3. ^*P<0.05, in comparison with the non-Cx45.6-overexpressing control. Bar, 10 µm.