Pgex 4t expression vector

For production of mouse antibodies, we designed two sets of primers. One pair amplified the Z-AR cDNA (AB372103) encoding polypeptide RVISCKRNNPASSSRRFFQL (AR20) corresponding to residues 695–714, and the other the CYP17A1 cDNA (AB284119) encoding polypeptide DEKEWVNPHLFNPDRFLDENGNRVYS (CYP1726), corresponding to residues 409–434. The DNA fragments were inserted into the BamHI/XhoI sites of the pGEX-4T expression vector (GE Healthcare). The GST-AR20 and GST-CYP1726 expression cassettes were transfected into E. coli BL21. The transfected cells were sonicated in ice-cold water using an Ultrasonic Processor (TAITEC, model VP-ST). We purified GST-AR20 and GST-CYP1726 proteins according to the manufacturer's protocol (GE Healthcare) and immunized 8-week-old BALB/c female mice three times with 50 µg of protein in complete Freund's adjuvant (Wako) at 2-week intervals. Sera were collected and tested 5 days after the last booster injection and used for immunohistochemistry.

The NuMA construct was a generous gift from Dr. Duane Compton, whereas p80 C-terminal constructs that contained the S535L and L540R mutations were generous gifts from Dr. Murat Gunel. The wild-type, truncated and mutated p80 were subsequently sub-cloned into a pEGFP vector (Clontech Laboratories, CA, USA). Recombinant p80 and NuMA44 (link) proteins were generated using a bacterial expression system (Invitrogen) and pGEX-4T expression vector (GE Healthcare Lifesciences, UK). Protein purification was performed using Glutathione Sepharose 4B (GE Healthcare Lifesciences, UK) according to the manufacturer’s instructions. The glutathione S-transferase (GST) tag was removed from recombinant proteins by thrombin treatment (GE Healthcare Lifesciences, UK).