Amylose resin

MBP fusion proteins were produced by expression of the appropriate pLEICS10-hSUN1 plasmids in E. coli BL21 and purification with amylose resin (New England Biolabs). For radiolabelled kinase assays, equal amounts (1–2 µg) of each MBP fusion protein, bound to amylose resin, or purified casein (Sigma) were incubated with 1 μCi of [γ-³²P]-ATP in kinase buffer (50 mM Hepes-KOH pH 7.4, 5 mM MgCl_2, 5 mM β-glycerophosphate, 5 mM NaF, 4 μM ATP, 1 mM DTT) and 5 μg of purified Cdk1/cyclin B or Plk1 (Merck Millipore) for 30 min at 30 °C. For non-radiolabelled kinase assays destined for MS analysis, [γ-³²P]-ATP was omitted and reactions were incubated for 4 h at 30 °C. Reactions were stopped by addition of 25 μl of Laemmli buffer and analyzed by SDS-PAGE and autoradiography or further processed for LC-MS/MS analysis.
Protein bands from the dried Coomassie-stained gels were excised and placed in scintillation vials along with OptiPhase HiSafe scintillation fluid (Perkin Elmer). Radioactivity was measured in counts per minute (cpm) on an LS 6500 multipurpose scintillation counter (Beckman Coulter). Percentage phosphorylation for each hSUN1 variant was calculated relative to casein.

The full‐length coding region of vemR was cloned into pET41a(+) at the BamHI and EcoRI sites, and that of rpoN2 was cloned into pMAl‐C4X at the BamHI and XhoI sites. The two constructs were transformed into BL21(DE3) cells for induction with 1.0 mm isopropyl‐β‐d‐thiogalactopyranoside (IPTG). Bacterial cells were harvested and resuspended in 10 mm phosphate‐buffered saline. Following sonication, insoluble cell debris was removed by centrifugation. GST‐VemR proteins were immobilized on a glutathione resin according to the manufacturer’s instructions (Genescript, Nanjing, China). MBP‐RpoN2 was purified by amylose affinity chromatography. The MBP pull‐down assay was performed using the method described previously (Liu et al. 2011). In brief, 3 µg of both MBP‐RpoN2 and GST‐VemR were incubated overnight at 4 °C with 300 μL of amylose resin (Sigma, Shanghai, China). The beads were collected by centrifugation and washed with 0.1% Triton X‐100 and increasing concentrations of NaCl to eliminate spurious protein interactions. Proteins were eluted from the amylose resin, boiled in 1 × SDS loading buffer and electrophoresed on a 12% sodium dodecylsulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) gel before being immunoblotted with anti‐MBP. A negative control was performed by incubation of 3 µg maltose binding protein (MBP) protein with GST‐VemR.