Mouse anti polyhistidine

SDS polyacrylamide gel electrophoresis was carried out under denaturing and reducing conditions using a Mini-PROTEAN III electrophoresis system following the manufacturer’s instructions (BioRad Laboratories, Mississauga, ON, Canada). Replica gels were transferred to nitrocellulose using an iBlot Gel Transfer Device as specified by the manufacturer (Invitrogen, Carlsbad, CA, USA). Immunoblotting was as previously described, using Tris-buffered saline/0.02% Tween 20 (TBST) for washing and 3% BSA in TBST for antibody incubations. Primary antibodies were all monoclonal: mouse anti-HSA (Genway Biotech, San Diego, CA; mouse anti-polyhistidine (Sigma-Aldrich, Oakville, ON, Canada); and mouse anti-myc (Thermo Fisher Scientific, Mississauga, ON, Canada). The secondary antibody was goat anti-mouse IgG, alkaline phosphatase- (AP-) conjugated. Blots were visualized through colour development resulting from the action of AP on substrate nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate, toluidine salt (NBT/BCIP).

The following reagents and antibodies were purchased commercially: mouse anti-Myc (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); goat anti-Ran GTPase (Santa Cruz Biotechnology Inc.); rabbit anti-tubulin (Sigma-Aldrich); rabbit anti-β-actin (Cell Signaling Technology, Beverly, MA, USA); rabbit anti-mouse immunoglobulins–horseradish peroxidase (IgG-HRP) (Dako, Glostrup, Denmark); swine anti-rabbit IgG-HRP (Dako); rabbit anti-goat IgG-HRP (Dako, Glostrup, Denmark); mouse anti-polyhistidine (Sigma-Aldrich); swine anti-rabbit immunoglobulins-fluorescein isothiocyanate (IgG-FITC) (Dako); rabbit anti-mouse IgG-FITC (Dako); clarity enhanced chemiluminescence solution (Bio-Rad, Hercules, CA, USA); and Hoechst 33258 (Molecular Probes, Invitrogen Corp., Carlsbad, CA, USA).