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Fig. 1. Expression and co-distribution of wild-type and mutant Cx45.6. (A) Recombinant retroviruses containing cDNAs encoding wild-type Cx45.6 (lane 3) and mutant Cx45.6(D47A) (lane 1) and Cx45.6(P88S) (lane 2) proteins were used to infect CEF cells. After 6 days of infection, cells were lysed, and crude membranes were prepared and analyzed by SDS-PAGE. Western blots were performed by probing PVDF replicas with affinity-purified antibody recognizing the C-terminus of Cx45.6. The membrane was stripped and re-probed with monoclonal antibody against the control protein, 
-actin. (B) Eight days after infection of recombinant retroviruses, primary cultured cells expressing exogenous wild-type Cx45.6 (a-c), mutant proteins Cx45.6(D47A) (e-g) and Cx45.6(P88S) (i-k) were fixed, stained with DAPI (a,e,i) and labeled with antibody against FLAG (b,f,j) or against Cx45.6 (c,g,k). The primary antibodies were detected by fluorescein-conjugated anti-mouse IgG for the antibody against FLAG and rhodamine-conjugated anti-rabbit IgG for the antibody against Cx45.6. The immunostaining was visualized by confocal fluorescence microscopy. The corresponding merged images derived from a,b,c, e,f,g and i,j,k are shown in d, h and l, respectively. Bar, 10 µm.
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