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Fig. 6. The induction of short processes depends on the interaction between UCH-L1 and monoubiquitin. (A) FLAG-tagged WT UCH-L1, C90S UCH-L1, D30A UCH-L1 and GFP (all in the pCI-neo vector) were transfected into NPCs. Antibodies against the FLAG-tag were used to detect transfected UCH-L1. The green staining shows transfected cells and the red staining shows endogenous nestin. Transient transfection of each construct was performed under proliferating conditions. At 48 hours after transfection, bFGF was removed for 12 hours before the cultures were immunostained. The lengths of nestin-positive processes in immunostained cells were measured. Asterisks indicate differences from the value of GFP-transfected NPCs at *P<0.05 and ***P<0.001. Bars, 80 µm. (B) Visualization of recombinant 6HN-tagged UCH-L1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie staining (B, left). UCH-L1 hydrolase activity was measured by Ub-AMC hydrolysis. Enzyme concentration was 4.3 nM, and substrate concentration was 700 nM. Initial velocity data was used to determine the values for relative hydrolase activity of UCH-L1 (B, right). (C) UCH-L1 co-immunoprecipitated with Ub. Cytosolic extracts from NIH-3T3 cell lines stably expressing HA-tagged WT UCH-L1 and mutants thereof were immunoprecipitated using anti-HA and immunoblotted with anti-HA antibody or anti-Ub antibody.
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