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Fig. 6. (A) Western blot of cells transfected with control or vimentin siRNA showing silencing of vimentin in TrHBMECs Extracts of TrHBMECs transfected with control (lane 1) or vimentin siRNA (lane 2) were processed for immunoblot analyses using antibodies against vimentin (upper panel). The blot was re-probed with actin antibody as a loading control (lower panel). Molecular masses are indicated on the left. (B) The immunoblots in A were scanned and analyzed using MetaMorph software. The amount of vimentin in each sample was then normalized to the actin control. Vimentin expression in the control siRNA-treated cells is represented as 100% (white bar). Vimentin expression in the vimentin siRNA-treated cells is reduced by about 60% (black bar). (C-H) Immunofluorescence analyses of cytoskeletal networks and ß3 integrin subunits in vimentin siRNA treated TrHBMECs. TrHBMECs were transfected with control siRNA or vimentin siRNA and then 72 hours later were prepared for double label immunofluorescence confocal microscopy using the combinations of antibody or cytoskeleton probes as indicated at the left. The merged images of the two staining patterns are shown in each case. Note that there is a dramatic perturbation in the vimentin cytoskeleton in D, F and H. The FCs labeled by the ß3 integrin antibody probe are considerably smaller in D than those labeled by the same probe in C (also see Fig. 9). The vimentin siRNA treatment has minimal, if any, impact on the organization of the microtubule or microfilament networks (compare F and H with E and G). Scale bar: 5 µm.
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