PMC:2728203 / 23626-27018
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"In vitro binding assays\nFor in vitro assays, purified recombinant Nab2-N (amino acids 1–97) and CT-Mlp1 (amino acids 1490–1779) were employed. Nab2-N was expressed as a tobacco etch virus protease-cleavable GST fusion protein to assess direct binding.37 GST-Nab2-N (pAC2058) was expressed in E. coli DE3 cells. Cells were collected and lysed in phosphate-buffered saline (PBS) (137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, pH 7.4) supplemented with protease inhibitor mixture (1 mM PMSF, 3 ng/ml pepstatin A, leupeptin, aprotinin, and chymostatin) by incubation with lysozyme (0.1 mg/ml) for 30 min on ice followed by sonication to purify the GST fusion proteins. Lysates were clarified by centrifugation and incubated with glutathione Sepharose (Amersham) in buffer A [20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM β-mercaptoethanol, 1 mM PMSF, and 0.1% Igepal] for 2 h at 4 °C with mixing. The beads were then washed with PBS and 0.5% Triton X-100.\nHis-CT-Mlp1 (pAC1486) was expressed in E. coli DE3 cells. Cells were collected and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 7.0) supplemented with protease inhibitor mixture by incubation with lysozyme and sonication. Lysates were clarified by centrifugation and incubated with Ni–NTA agarose (Qiagen) in lysis buffer for 2 h at 4 °C with mixing. The beads were then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole). His-Nab2 and His-CT-Mlp1 were eluted from agarose with 250 mM imidazole. Sepharose-bound GST or GST-CT-Mlp1 (6 μg) was incubated with 2 μg of purified His-Nab2 at 4 °C in PBS for 90 min. Sepharose-bound GST-NT-Nab2 WT, F72D, or F73D (6 μg) was incubated with purified His-CT-Mlp1 (2 μg). Unbound fractions were collected and the beads were washed three times with PBS. Bound fractions were eluted with sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue) and analyzed by SDS-PAGE followed by Coomassie Blue staining.\nEach GST-Nab2-N fusion protein or the GST control protein was added to yeast lysate to examine the interaction between the N-terminal domain of Nab2 and full-length Mlp1. GST fusion proteins were then purified on glutathione beads, and copurifying proteins were visualized either by Coomassie Blue staining or by immunoblotting.\nPurified recombinant His-tagged Gfd1 was prepared as previously described to examine the interaction between Nab2-N and Gfd1.27 The Gfd1 protein was attached to CNBr Sepharose beads as previously described.38 Briefly, CNBr Sepharose beads (Amersham Pharmacia Biotech) were swollen and washed in 1 mM HCl. Beads were transferred to coupling buffer (100 mM NaHCO3, pH 8.3, and 500 mM NaCl) and added to 2–5 mg of Gfd1 in coupling buffer. Coupling was carried out at 4 °C overnight. Residual active groups were blocked with 1 M Tris–HCl, pH 8.0, for 2 h at room temperature. Beads were then washed successively and extensively four times in coupling buffer and acid wash buffer (0.1 M sodium acetate, pH 4.0, and 500 mM NaCl). For binding assays, 10 μg of Nab2-N was incubated with 50 μl of Gfd1 beads for 2 h at 4 °C. Beads were then washed twice in PBS, and bound proteins were eluted with 100 μl of sample buffer. Samples were resolved by PAGE, and bound proteins were detected by Coomassie Blue staining.","divisions":[{"label":"title","span":{"begin":0,"end":23}},{"label":"p","span":{"begin":24,"end":992}},{"label":"p","span":{"begin":993,"end":2057}},{"label":"p","span":{"begin":2058,"end":2386}}],"tracks":[{"project":"2_test","denotations":[{"id":"18190927-15208322-62517995","span":{"begin":2512,"end":2514},"obj":"15208322"},{"id":"18190927-8918934-62517996","span":{"begin":2593,"end":2595},"obj":"8918934"}],"attributes":[{"subj":"18190927-15208322-62517995","pred":"source","obj":"2_test"},{"subj":"18190927-8918934-62517996","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#aaec93","default":true}]}]}}