Ab45938

TPX2 antibody was a kind gift from Oliver Gruss24 (link). Anti-RanGTP antibody (AR-12) was a kind gift from Ian Macara37 (link). Mouse anti-α-tubulin monoclonal antibody DM1A (T6199) and Duolink reagents were from Sigma (St Louis, MO). Mouse anti-β-III-tubulin antibody TUJ1 (MMS-435P) and mouse anti-neurofilament monoclonal antibody SMI312 (SMI-312R) were from Covance (Princeton, NJ). Mouse anti-γ-tubulin antibody GTU-88 (GTX11316), HRP-labeled goat-anti-rabbit IgG antibody (GTX213110), and HRP-labeled goat-anti-mouse IgG antibody (GTX213111) were from GeneTex (Irvine, CA). Mouse anti-actin antibody C4 (MAB1501), mouse anti-GAPDH antibody 6C5 (MAB374), and rabbit anti-MAP2 polyclonal antibody (AB5622) were from Millipore (Billerica, MA). Rabbit anti-Ran polyclonal antibody (ab31118) and rabbit anti-importin β1 (ab45938) were from Abcam (Cambridge, UK). Mouse anti-importin-α antibody (sc-55538) was from Santa Cruz Biotechnology (Santa Cruz, CA). Dylight-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, PA). Alexa Fluor-conjugated secondary antibodies were from Life Technologies (Carlsbad, CA).

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).