Nb500 186

HeLa, A431 (both purchased from ATCC), and Mel 624 melanoma cells (gift from Suzanne Topalian, Johns Hopkins University) were cultured using standard tissue culture procedures. For the immunoblots shown in Figure 3B, A431 and HeLa cells were washed extensively with PBS before lysing with buffer A (1% Nonidet P-40, 20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, and a protease inhibitor cocktail). Cell lysates (20 μg/lane for the TIF1-γ blots and 5 μg/lane for the CCAR1 blots) were electrophoresed in 10% SDS-PAGE gels and transferred to nitrocellulose membranes. Immunoblots were performed using a rabbit polyclonal anti-CCAR1 antibody (Novus Biologicals, NB500-186; 1:7,500 dilution) or a mouse monoclonal anti–TIF1-γ antibody (Novus Biologicals, H00051592, clone 6D1; 1:1,000 dilution), followed by incubation with horseradish peroxidase–labeled secondary antibodies (Pierce) and chemiluminescence. Images were acquired using a Protein Simple Fluorochem-M digital imager. The same antibodies were used for blotting the immunoprecipitations shown in Figure 3C and Supplemental Figure 2.

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).