Figure 3
A – In vitro translation of Parvulin isoforms. Open reading frames for Par17-QR, Par17-RS and Par14 were TOPO-cloned into the vector pCR-4 and 35S-methionine labeled in an in vitro transcription/translation reaction. Reaction products were separated by 17.5% SDS-PAGE following autoradiography. B – Proteinase K digestion of Parvulin isoforms. Equal amounts of 35S-methionine labeled Par17-QR, Par17-RS and Par14 were incubated with increasing concentrations of proteinaseK (PK) for 30 min at 10°C. Enzyme concentrations are given in μg/ml. Reactions were stopped by an excess of PMSF, separated by SDS-PAGE and subjected to autoradiography. C – Western blots of cell lysates. Western blot of HepG2 (lane 1 and 3) and HeLa (lane 2 and 4) cell lysates. Proteins were separated by SDS-PAGE with MES as running buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes. Blots were incubated with pre-immune (lane 1 and 2) or anti-Par17 serum (lane 3 and 4; both at 500-fold dilution). D – The newly raised antibody Ab-EXT recognizes Par17-RS and -QR. Coding sequences for Par17-RS and -QR were subcloned in the pET-28 vector with N-terminal His6 tag and expressed in E. coli. Lysates were subjected to reducing SDS-PAGE in MES buffer and SeeBlue2 as protein standard, transferred to nitrocellulose membranes and incubated with the anti-Par17 antibody. -, before IPTG induction; RS, Par17-RS lysates; QR, Par17-QR lysates. Coomassie stained gel of lysates to show equal loading. Par17 fusions with His6 tag and thrombin cleavage sites show apparent molecular weights of about 22 KDa in SDS-PAGE. The induction band in E. coli lysates and the corresponding band recognized by Ab-EXT are labeled with arrows. The lower migrating band may be caused by proteolytic degradation. E – Ab-EXT does not recognize GFP-Par14. Par14 coding sequence was expressed as GFP fusion in HeLa cells (lane 3). Lane 1 and 2 are HeLa cell lysates not transfected with this construct. Lysates were subjected to reducing SDS-PAGE in Tris-glycine buffer with MagicMark as protein standard, transferred to nitrocellulose membranes and incubated with anti-Par17 and anti-GFP antibodies. Coomassie stained gel of lysates to show equal loading.