Recombinant proteins containing a hexahistidine (His6) or glutathione S-transferase (GST) tag were expressed in Escherichia coli BL21 (DE3) and then isolated using Ni2+-nitrilotriacetic acid resin (Qiagen, Valencia, CA) or GSH-Sepharose (GE Healthcare, Chalfont St. Giles, Buckinghamshire, United Kingdom), respectively. After elution, the proteins were dialyzed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM ethylene glycol tetraacetic acid (EGTA), and 10% glycerol and then stored at −80°C.
Two anti-NME7 antisera were generated by immunizing rabbits with NME7 1–140 and full-length NME7 prepared as His6-tagged proteins. The sera obtained were named sera 651 and 673, respectively. Both antibodies were purified using respective antigens in fusion with GST and immobilized on polyvinylidene fluoride membranes. The two antibodies gave similar results in experiments, and most of the data presented here were collected using antibody 651. The production of the following antibodies has been described previously: rabbit anti-CDK5RAP2, anti-GCP2, anti-GCP3, anti-GCP4, anti-GCP5, and anti-GCP6 (Fong et al., 2008 (link); Choi et al., 2010 (link)). These mouse monoclonal antibodies were purchased from Sigma (St. Louis, MO): anti-FLAG (M2), anti–γ-tubulin (GTU-88), anti–α-tubulin (DM1A), and anti–β-actin (AC-15).
Two anti-NME7 antisera were generated by immunizing rabbits with NME7 1–140 and full-length NME7 prepared as His6-tagged proteins. The sera obtained were named sera 651 and 673, respectively. Both antibodies were purified using respective antigens in fusion with GST and immobilized on polyvinylidene fluoride membranes. The two antibodies gave similar results in experiments, and most of the data presented here were collected using antibody 651. The production of the following antibodies has been described previously: rabbit anti-CDK5RAP2, anti-GCP2, anti-GCP3, anti-GCP4, anti-GCP5, and anti-GCP6 (Fong et al., 2008 (link); Choi et al., 2010 (link)). These mouse monoclonal antibodies were purchased from Sigma (St. Louis, MO): anti-FLAG (M2), anti–γ-tubulin (GTU-88), anti–α-tubulin (DM1A), and anti–β-actin (AC-15).
