Mapping of Ras-related GTP-binding proteins by GTP overlay following two-dimensional gel electrophoresis.

Abstract

For identification of Rab, Rac, Rho, Ral, Rap, and Arfproteins on two-dimensional polyacrylamide gels, we have expressed full-length cDNAs of members of these protein families with the T7 RNA polymerase-recombinant vaccinia virus expression system. Membrane preparations from cells expressing the cDNAs were subjected to highresolution two-dimensional polyacrylamide gel electrophoresis followed by [a-32P] (17,19). Primary cultures of rat hippocampal neurons were prepared as described (20). Cells were cultured for 9 days (stage 5 cells) before use (21).Infection with T7 polymerase-recombinant vaccinia virus (22) was carried out in serum-free medium (17). After 30 min cells were supplemented with 5% serum and transfected by using N- [1-(2,3-dioleoyloxy)propyl]-N,NN-trimethylammonium methyl sulfate (DOTAP) (Boehringer Mannheim) according to the manufacturer's instructions and incubated in the presence of 10 mM hydroxyurea for 5 hr at 370C in a 5% CO2 atmosphere.Preparation of Total Cell Membrane Fractions and Synaptosomes. A postnuclear supernatant (17) was transferred to a Beckman TLA100.2 microcentrifuge tube and centrifuged at 60,000 rpm for 30 min at 40C. The supernatant (cytosol fraction) and the pellet were separated and processed for analysis. A fraction enriched in synaptosomes (18) was isolated from 15 rat brains. Two-Dimensional PAGE and Transfer to Nitrocellulose for [e-32P]GTP Overlay and Immunoblotting. A combination of isoelectric focusing (IEF) and SDS/PAGE was used to resolve proteins in two dimensions essentially as described previously (17). For transfer to nitrocellulose for GTP overlay and immunoblotting we have modified a protocol (17) Abbreviations: IEF, isoelectric focusing; GDI, GDP dissociation inhibitor; GST, glutathione S-transferase. tPresent address:

The role of CAP3 in CD95 signaling: new insights into the mechanism of procaspase-8 activation

“…N1-Sepharose beads (30 ml) were used for immunoprecipitation at 41C for 2 h. All beads were washed five times with 20 volumes of lysis buffer. The immunoprecipitates were analyzed either using 2-D isoelectric focusing (IEF)/SDS-PAGE as described 30 or 1-D gels. The gels were transferred to Hybond nitrocellulose membrane (Amersham Pharmacia Biotech, Germany), blocked with 5% nonfat dry milk in PBS/Tween (PBS plus 0.05% Tween 20) for 1 h, washed with PBS/Tween, and incubated with the primary antibody in PBS/Tween at 41C overnight.…”

Nucleotide induced conformation determines posttranslational isoprenylation of the ras related rab6 protein in insect cells

“…Secondary goat anti-mouse IgG coupled to peroxidase and [~-32P]GTP (3000 Ci/mmol) were from Amersham (Braunschweig, Germany) and [3H]mevalonolactone (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Ci/mmol) from DupontNEN (Bad Homburg, Germany). Reticulocyte lysate was obtained from Serva (Heidelberg, Germany).…”

A novel approach for expression cloning of small GTPases: identification, tissue distribution and chromosome mapping of the human homolog of rheb

“…[9][10][11]. By using this assay we have detected more than 40 low molecular weight GTP-binding proteins in normal human keratinocytes, determined their relative abundance in normal proliferating and SV40 transformed keratinocytes (K 14), and identified several unknown GTPases that are strongly down-regulated in the transformed cells and that may be required for the maintenance of the normal phenotype [10].…”

“…By using this assay we have detected more than 40 low molecular weight GTP-binding proteins in normal human keratinocytes, determined their relative abundance in normal proliferating and SV40 transformed keratinocytes (K 14), and identified several unknown GTPases that are strongly down-regulated in the transformed cells and that may be required for the maintenance of the normal phenotype [10]. Based on the same approach, a 2D gel database of many cloned ras-like GTPases has been established by Huber et al [11]. The number of small GTPases that can be revealed by this technique is limited however by the low abundancy of these proteins.…”

“…To overcome this problem, we thought to take advantage of the fact that small GTPases comprising the ras superfamily are able to bind GTP following denaturation with sodium dodecyl sulfate (SDS) and transfer to nitrocellulose membranes [9,11,13]. Based on the above premises we have developed a novel and simple approach for the identification and cloning of low molecular weight GTP-binding proteins.…”