Cell-cell adhesion regulates Merlin/NF2 interaction with the PAF complex
Fig 4
CDC73 interacts with VPRBP and is ubiquitylated in vivo.
(A) 293T cells were transfected with HA-ubiquitin, indicated Flag-proteins and either empty vector or VPRBP. Flag IPs were immunoblotted as indicated. The asterisk points to a band corresponding to Flag-AMOTb bait that crossreacts with the HA-antibody. (B) 293T cells were transfected as above with but using TAP6-CDC73 (containing a tandem His tag and Flag tag) and lysed and purified under denaturing condition. Talon beads pull-downs were immunoblotted as indicated. Asterisk points to a band corresponding to the TAP6-CDC73 bait that cross-reacts with the HA antibody on lycor. (C) VPRBP/DCAF1 but not other DCAFs stimulate CDC73 ubiquitination. As in A but comparing Myc-VPRPB to other Myc-DCAFs. (D) Merlin partially inhibits VRPBP stimulated ubiquitylation of CDC73. Flag-CDC73 was transfected with HA-UB and either Merlin or Ezrin as control as in A. Ubiquitin associated with Flag-IPs was detected by wb with an HA antibody. (E) Endogenous CDC73 co-IPs with VPRBP in multiple cell lines. WB: Western blot, IP: Immunoprecipitation, PD: Pull down.