Figure 4
Inhibition of Bmp2 proprotein processing does not increase nuclear localization. (a) In vitro synthesis of radiolabeled Bmp2 preproprotein produced a 43 kDa protein (lane 1, large arrow). Incubation of this protein with recombinant furin for 1 or 3 hours generated a new protein band at 31 kDa, the predicted size of the free Bmp2 propeptide following proteolytic cleavage (lanes 2 and 3, small arrow). Preincubation of the furin with α1PDX, a serine protease inhibitor that blocks furin activity, prevented the formation of this band (lane 4). (b) Furin and α1PDX expression plasmids were each cotransfected with the wtBmp2/GFP fusion plasmid into RCS cells. Increasing furin expression did not significantly decrease nuclear localization of Bmp2/GFP, nor did inhibiting furin activity with α1PDX significantly increase nuclear localization. Mutation of the Bmp2 cleavage site to make it unrecognizable by furin or any related proprotein convertase (mtBmp2/GFP) also failed to significantly increase nuclear localization of Bmp2/GFP.