Nup358

Protein extracts were treated as previously described [5 (link)]. Briefly, between 25 and 40μg of proteins were separated by SDS-PAGE (8%, 10% or 12% acrylamide), and transferred to PVDF membranes. Membranes were blocked in Tris Buffered Saline and tween 0.1% (TBS-T) containing 5% BSA for 1 hr and incubated either 2 hrs at room temperature or over-night at 4°C with primary antibody. After washing with TBS-T (2 times for 5min), membranes were incubated 1 hr with secondary anti-HRP-conjugated antibody (GE Healthcare, Mississauga, ON, Canada). After washing with TBS-T (3 times for 5min), they were developed by chemoluminescence immunodetection with ECL reagents (Thermo Fisher Scientific, Rockford, IL) and autoradiography.
List of antibodies used: GP63 monoclonal antibody clone #253 (Button et al. 1991); Histone H2B (Genscript corporation, A01174); KDEL (ab12223), SHP-1 (ab3254), NPC Mab414 (ab24609), Nup62 (ab96134), Nup358 (RanBP2, ab64276), KpnB1 (ab2811), KpnA3 (ab105348) from Abcam; PGK1 (ProteinTech, 17811-1-AP); Actin (Sigma, A5316); TCPTP (MediMabs, MM-0018-P); NF-κB p65 (SC-8008), Nup93 (SC-374400), Nup214 (SC-26055), RanGAP1 (SC-1862), Kif1B (SC-28540) from Santa Cruz; C-Jun (Cell Signaling, 60A8).

Cell lysates were harvested and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western Blot analysis using the Odyssey System (Li-Cor) as previously described [78 (link)]. Antibodies were obtained for specific detection of Nup358 (Abcam), LEDGF/p75 (Cell Signaling), or α-tubulin (Sigma). Primary antibodies were detected using either infrared dye-labeled (800C) goat anti-rabbit or infrared dye-labeled (680) goat anti-mouse secondary antibodies (Li-Cor).