Fig. 4.

A LMP-1 specifically interacts with Jab1. LMP-1 is associated with a 37-kDa protein. Recombinant LMP-1 was labeled with sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3-dithiopropionate-biotin transfer reagent and incubated with nuclear lysates of hMSC cells. Biotin transfer to interacting proteins was accomplished as described under “Materials and methods.” Biotinylated proteins were enriched using neutravidin beads, separated by SDS-PAGE, and detected on western blots using HRP-labeled neutravidin and ECL. Bands were excised for tryptic digestion and NanoLC-MS/MS analysis. B The identity of Jab1 was confirmed in western blots using Smurf-1 and Jab1-specific antibodies on immunoprecipitates obtained by antibiotin antibody. Western blots show the presence of both Smurf1 and Jab1 with neutravidin-horse radish peroxidase (lane 1), Smurf1 with Smurf1 antibody (lane 2) and Jab1 with Jab1 antibody (lane 3). The 85 and 37 kDa bands were identified as Smurf1 and Jab1, respectively, as shown by western blotting