FIG. 5.

Dominant-negative dynamin 2ab functions downstream of type I TGF-βR phosphorylation. R1B cells were transiently transfected with the native HA-tagged type I TGF-βR (TGFβ-RI) and either GFP-tagged wild-type (Dyn Wt) or dominant-negative (Dyn KA) dynamin 2ab isoforms. (Top) After in vivo labeling with ³²P, cells were treated as indicated with 15 ng of TGF-β/ml for 25 min. Normalized protein lysates were immunoprecipitated by using anti-HA affinity matrix and processed as described in Material and Methods. (Middle and bottom) Parallel plates were treated identically to those in the top panel but without orthophosphate labeling to verify transfected dynamin (middle panel) and type I receptor (bottom panel) expression. Equivalent protein lysates were processed for Western blotting with antisera to GFP (Dyn-GFP) or the type I TGF-βR (TIR).