Generation and Characterization of RanBPM Mutants

pCMV-HA-RanBPM and deletion mutants have been previously described [34 (link),36 (link)]. pEBG-GST-ΔN-c-Raf was a gift from Dr. Zhijun Luo (Boston University, Boston, MA, USA). pET28a-ΔN-c-Raf was generated by isolating ΔN-c-Raf from pEBG-GST-ΔN-c-Raf and subcloning into pET28a (EMD Millipore, Billerica, MA, USA). pBSK-HA-Ubiquitin was a gift from Dr. Lina Dagnino (Western University, London, ON, Canada). pCGN-HA-RMND5A was obtained by subcloning RMND5A cDNA obtained by RT-PCR from a Jurkat T cell cDNA library into pCGN-HA plasmid. All pGEX4T1-GST-RanBPM wild-type (WT) and mutant constructs were generated by polymerase chain reaction (PCR) and subcloned in the pGEX4T1 vector (GE Healthcare Life Sciences, Little Chalfont, UK). RanBPM point mutants (pGEX-4T-1-GST-C4 Q703L T750L and pCMV-HA-RanBPM R625L E626L) were generated by site-directed mutagenesis. All PCR reactions were performed using KOD Hot Start Polymerase PCR kit (EMD Millipore).