FIGURE 9.

Depletion of VAP-A results in an abnormal subcellular distribution of protrudin. A, PC12 cells were transfected for 48 h with an expression vector for VAP-A-shRNA 2 or VAP-B-shRNA 2 or with the corresponding empty vector (pIRES-Venus-B). The cell extracts were then prepared and subjected to immunoblot (IB) analysis with anti-VAP-A, anti-VAP-B, or anti-calnexin. B, PC12 cells transfected as in A were stimulated with NGF for 6 h and then subjected to immunofluorescence analysis with anti-protrudin (red) or anti-PDI (red). Arrowheads indicate the pericentrosomal region. C, PC12 cells transfected as in A but in the additional presence of an expression vector for 3× FLAG-protrudin were stimulated with NGF for the indicated times and then subjected to immunofluorescence staining with anti-FLAG (red). The cells were also monitored for Venus fluorescence (green). D, HeLa cells were transfected for 48 h with an expression vector for VAP-A-shRNA 3 or for an shRNA specific for human VAP-B (or with the corresponding empty vector, pIRES-Venus-B) as well as with a vector for 3× FLAG-protrudin, after which cell extracts were subjected to immunoblot analysis with anti-VAP-A, anti-VAP-B, or anti-calnexin. E, HeLa cells transfected as in D were stained with anti-FLAG (red) or anti-PDI (red). The cells were also monitored for Venus fluorescence (green). Arrows indicate cells depleted of VAP-A (Venus positive); the arrowhead indicates a cell not depleted of VAP-A (Venus negative). The merged images are also shown. F, a model of VAP-A function in the regulation of protrudin.