Pet28a

pET28a-CDKG1 or pET28a-CDKG1∆N (missing residues 1–92) were made by PCR amplification from pGEM-T-CDKG1 using primer sets described in Supplementary file 2 and ligation into pET28a (EMD Millipore, Billerica, MA) at NdeI/EcoRI sites. pGST-MAT3 was generated as described previously (Olson et al., 2010 (link)). All recombinant proteins were expressed using E.coli BL21 codon plus-RIL strain (Agilent Technologies). Purification of insoluble 6xHis-CDKG1 and 6xHis-CDKG1∆N was performed under denaturing conditions as described previously (Olson et al., 2010 (link)). Purified 6xHis-CDKG1 was cut out from a Coomassie blue stained SDS-PAGE gel and sent to Cocalico Biological Inc. (Reamstown, PA) to generate rabbit polyclonal anti-sera. Polyclonal antibodies raised against CDKG1 were affinity purified with AminoLink Plus Resin (Pierce Thermo Fisher Scientific, Waltham, MA) coupled to purified His-CDKG1∆N as described previously (Olson et al., 2010 (link)).

For the recombinant protein expression, the CDS of NF‐YB1 and NF‐YC12 were amplified and cloned into pET28a (Merck, Darmstadt, Germany) and pGEX‐4T‐1 (GE Healthcare, Chicago, IL) respectively. For HIS‐bHLH144‐FLAG construct, FLAG sequence was synthesized on the reverse primer, and then the amplicon was cloned into the pET28a (Merck, Darmstadt, Germany).
HIS‐NF‐YB1, GST‐NF‐YC12, HIS‐bHLH144‐FLAG recombinant proteins were induced in E. coli strain Rossetta, and purified by Glutathione‐Sepharose Resin Protein Purification Kit and 6 X His‐Tagged Protein Purification Kit (CWBIO, Beijing, China) respectively. Pull‐down assay was conducted as following: 50 μL equilibrated Glutathione High Capacity Magnetic Agarose Beads (Sigma, St Louis, MO) or anti‐FLAG M2 Magnetic beads (Cat No. M8823, Sigma‐Aldrich, St. Louis, MO) was mixed with 500 μg of each recombinant protein in 600 μL pull‐down buffer (50 mm Tris‐HCl pH = 7.5, 5% glycerol, 1 mm EDTA, 1 mm DTT, 1 mm PMSF, 0.01% Nonidet P‐40, 150 mm KCl) under 4 °C for 2 h. The bound proteins together with the beads were collected, washed with pull‐down buffer twice, eluted with 50 μL 1× PBS and immune detected by GST (Cat: CW0085, CWBIO, Beijing, China), HIS (Cat: CW0083, CWBIO, Beijing, China) and FLAG (Cat: CW0287, CWBIO, Beijing, China) antibodies respectively.

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).

The Tde-SAS (TDE_RS08190, formerly TDE1711) and Tde-SAH (TDE_RS08100, formerly TDE1690) genes were PCR amplified from T. denticola ATCC 35405^T genomic DNA and cloned into pET28a (+) (Novagen, Merck Millipore) via BamHI/XhoI to create plasmids pET28a-Tde-SAS and pET28a-Tde-SAH, respectively (Table S1). The catalytic domain (Tde-SAS_1–246) of Tde-SAS was analogously cloned to create plasmid pET28a-Tde-SAS_1–246. The FN0926 gene from Fusobacterium nucleatum ATCC 25586^T was analogously cloned into pET28a to create plasmid pET28-Fn-SAS. The Tde-SAH_D74A mutant was created using the Phusion site-directed mutagenesis kit (Thermo Fisher Scientific, USA). The Tde-SAH and Tde-SAH_D74A genes were respectively subcloned into pGEX-4T1 expression vectors via BamHI/XhoI to create plasmids pGEX-Tde-SAH and pGEX-Tde-SAH_D74A. The genes were analogously PCR amplified and cloned into pBAD33 plasmids (67 (link)). PCR primer details are shown in Table S2. Plasmid integrity was confirmed by Sanger sequencing. The plasmids were routinely maintained in E. coli DH10B (Invitrogen) and cultivated in Luria Bertani (LB) or LB-agar media (USB) containing kanamycin (50 μg/mL).