Proteomics

Immunoprecipitated proteins were eluted by incubation in 30 μl of denaturation buffer (6 M urea, 2 M thiourea, 10 mM Tris–HCl, pH 8.0) for 5 min at room temperature, and the entire eluate processed for mass spectrometry (without quantification). Proteins were reduced by incubation with dithiothreitol at a final concentration of 1 mM at room temperature (RT) for 1 h and free cysteine residues alkylated by incubation with iodoacetamide (Amresco Biochemicals and Life Science products) at a final concentration of 5.5 mM for 1 h at room temperature in the dark. The samples were diluted with 4 volumes of 20 mM ammonium bicarbonate (Sigma Aldrich) and 20 mM calcium chloride (Sigma Aldrich). Sequence-grade trypsin (New England Biolabs) was added to the samples with a protein to trypsin ratio of 50:1 and the samples were incubated for digestion at RT overnight. The digestion was stopped by addition of formic acid at a final concentration of 0.1%. Digested peptides were desalted using homemade STAGE tips with Empore™ Octadecyl solid-phase extraction disks (Supelco). STAGE tips were activated by adding 300 μl of solvent B (80% acetonitrile and 0.1% formic acid) and equilibrated by adding twice 100 μl of solvent A (2% acetonitrile and 0.1% formic acid). The samples were added to the STAGE tips and washed three times with solvent A. The bound peptides were then eluted three times by addition of 50 μl of solvent C (60% acetonitrile and 0.1% formic acid). The eluted peptides were dried in vacuo and resuspended in 50 μl of solvent A prior to measurement on a Q Exactive Mass Spectrometer (Thermo Fisher).

Tryptic peptides were separated by liquid chromatography on a homemade precolumn (100 μm ID × 20 mm) packed with C18 Luna beads (5 μm diameter, 100 Å pore size; Phenomenex 04A-5452) connected to an analytical column (75 μm × 200 mm) packed with Aeris C18 beads (3.6 µm diameter; Phenomenex 00B-4507-AN) connected to an Ultimate 3500 RS nano UPLC system (Dionex). Desalted peptides were loaded onto the column with a starting mobile phase of 2% ACN with 0.1% formic acid and separated at a constant flow rate of 300 nL/min using the following gradient: increase to 5% ACN over 5 min, increase to 50% ACN over 15 min, to 80% ACN over 5 min, followed by a column wash of 80% for 20 min. Mass spectra were collected on a Q Exactive mass spectrometer (Thermo Fisher Scientific) operated in a data-dependent manner with automatically switching between MS and MS/MS scans using a top-10 method. Peptides were ionised by electrospray ionisation and MS spectra were acquired at a resolution of 70,000 with a target value of 3 × 10⁶ ions or a maximum integration time of 250 ms. The scan range was restricted between 300 and 1750 m/z. Peptide fragmentation was performed by higher-energy collision dissociation (HCD) with the energy set at 25 NCE. Intensity threshold for ions selection was fixed at 1.7 × 10⁴ with charge exclusion of z = 1 and z > 5. The MS/MS spectra were acquired at a resolution of 17,500, with a target value of 2 × 10⁵ ions or a maximum integration time of 120 ms and the isolation window was set at 4.0 m/z.

Immunoprecipitated protein (the entire immunoprecipitate from an independent IP experiment to that performed for mass spectrometry) was resuspended in 35 μl of 2 × loading buffer without bromophenol blue. Samples were boiled at 95 °C for 5 min and centrifuged at 18,000×g for 3 min at RT and bromophenol blue then added to the supernatants. 35 μl sample was electrophoresed for Western blot analysis, performed using rabbit anti-Importin beta (1:5000, Abcam ab45938), rabbit anti-CRM1 (H-300) (1:1000, Santa Cruz Biotechnology, sc-5595), rabbit anti-Kpnα2 (1:2500, Abcam ab97580), mouse anti-Ran (1:500, Sigma-Aldrich R4777), rabbit anti-CCAR1 (1:1000, Novus Biologicals NB500-186), rabbit anti-FUBP1 (1:500, Novus Biologicals NBP2-16543) and mouse anti-GAPDH (0411) (1:10,000, Santa Cruz Biotechnology, sc-47724). For all IP-WB analysis, Abcam Veriblot for IP Detection Reagent (HRP) (ab131366) was used as a secondary antibody with a dilution of 1:2500 in 5% milk in TBST. For analysis of whole cell lysates, 30 µg protein was loaded onto SDS-PAGE gels and blots probed using the same antibodies mentioned above. Lumiglo (KPL) was used as the chemiluminescent substrate for Western blot detection. For all Western blot analyses, membranes were cut prior to hybridisation with antibodies. Images of the original blots can be seen in the supplementary material (Supplementary fig. ^S11–^S17).