PMC:1887720 / 12764-14357 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1887720","sourcedb":"PMC","sourceid":"1887720","source_url":"https://www.ncbi.nlm.nih.gov/pmc/1887720","text":"Generation of Fusion Proteins and In Vitro Binding Assays.\nFusion proteins were derived by transforming Escherichia coli with pGEX2T vectors containing full-length PSTPIP1, PTP-PEST, WASp, or cdc42 cDNAs, or PCR-amplified fragments representing the PSTPIP1 coiled coil (amino acids 120–358; PSTPIPCOIL) or SH3 (amino acids 365–415; PSTPIPSH3) domains, or pQE-30 vectors (QIAGEN) containing the Fyn, PST-PEST, or PSTPIP1 cDNAs. Fusion proteins were purified from isopropyl-1-thio-β-D-galactopyranoside–induced bacteria using glutathione-coupled sepharose 4B or Ni-NTA agarose beads (QIAGEN), and the amount of bound protein was estimated by Coomassie staining. For binding studies, 5 μg immobilized gluthathione S-transferase (GST) fusion proteins were incubated for 2 h at 4°C with either 250 μM purified 6XHis-tagged protein or lysates from stimulated Jurkat E6 cells. The immune complexes were resolved by SDS-PAGE and immunoblotting analysis was performed with the indicated antibodies. To assay WASp-cdc42 binding, 100 μg GST-cdc42 or GST alone immobilized on glutathione sepharose 4B beads was incubated for 10 min at 30°C with 50 μl GTPγS loading buffer (50 mM Tris-HCl, pH 7.5, 5 mM EDTA, 10 mM GTPγS) followed by the addition of 10 mM MgCl2. Beads were then incubated in 5 mM MgCl2 for 2 h at 4°C with 0.5 mg lysates obtained from Cos-7 cells 36 h after Lipofectamine-mediated transfection of pcDNA3.1 vectors encoding either WASp or WASpΔGBD. Complexes were washed and resolved by SDS-PAGE and sequential immunoblotting analysis was performed with anti-WASp and anti-cdc42 antibodies.","divisions":[{"label":"Title","span":{"begin":0,"end":58}}],"tracks":[]}