Anti ran

For western blot analysis, proteins were detected using the following antibodies: anti-TAZ (BD Biosciences, 560235, 1:1000), anti-RAN (Cell Signaling Technology, #4462, 1:1000), anti-c-Myc (Santa Cruz Biotechnology, SC-40, 1:1000), anti-pan-14–3–3 (Santa Cruz Biotechnology, SC-629, 1:1000), and anti-GFP (SC-8334, 1:1000). Jackson ImmunoResearch Laboratories was the source for HRP, IRDye680RD, and IRDye800CW conjugated secondary antibodies. Alexa 488 or 555 conjugated secondary antibodies were from Invitrogen. HRP conjugated secondary antibodies were used in 1:5000 for western blot analysis, fluorescent secondary antibodies 1:10000. Leptomycin B, Rapamycin, and okadaic acid were purchased from Sigma Aldrich and RhoII activator from Cytoskeleton Inc. Phos-tag gels (Wako Pure Chemical Industries, Ltd.) were ordered from Cedarlane.

Total cellular proteins were harvested and separated by SDS‐PAGE gels and then transferred onto PVDF membranes (Millipore). Equal protein loading was verified by the Bradford assay. The primary antibodies used in Western blot were the same as those in ‘Immunocytochemistry’. Membranes were blocked for 30 min at room temperature with 5% non‐fat dry milk in TBS containing 0.05% Tween‐20 (TBST), probed with the primary antibodies at a 1:1000 dilution for 1 h at room temperature and washed four times with TBST. The secondary anti‐mouse/rabbit IgG‐HRP (DAKO) was added for 1 h at room temperature. After extensive rinsing, immunoreactive protein bands were visualized with the ECL detection system (Santa Cruz, USA) and subsequently exposed to film. Densitometric assay was performed using Quantity one software (Bio‐Rad, USA). The specific primary antibodies were purchased from the following resource: anti‐FLAG (Sigma), anti‐MCM3 (Cell Signaling Technology, California, USA), anti‐NPM1 (Sigma), anti‐RAN (Cell Signaling Technology, California, USA), anti‐YBX1 (Abcam, Cambridge, UK), anti‐GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA) and anti‐KPNA2 (Abcam, Cambridge, UK).