Tnt reticulocyte lysate system

Glutathione-S-transferase (GST) fusion proteins were prepared as previously described [22 (link)]. The GST-E47bHLH expression plasmid was generated by PCR. GST-PU.1, GST-E47bHLH, or GST bound to glutathione agarose were incubated with ³⁵S-methionine labeled in vitro translated protein in 500 μL of NETN buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, and 0.5% NP-40). In vitro translated products were prepared by the TNT reticulocyte lysate system (Promega). After 4 h incubation bound complexes were washed 4x in NETN buffer and eluted in sample buffer followed by separation by SDS-PAGE. Plasmids containing PU.1 deletion mutants and full-length Pax5 were obtained from Dr. M. Atchison (University of Pennsylvania) [20 (link), 23 ]. Full-length human E47 was in vitro translated from the T7-E2/5 plasmid supplied by Dr. T. Kadesch (University of Pennsylvania). Truncations CT1 and CT2 were generated by linearizing T7-E2/5 plasmid with NotI and XhoI, respectively, before in vitro transcription/translation reaction. The amino terminal E47 truncations were generated by PCR. Human EBF protein was in vitro translated from pSport-EBF obtained from ATCC. ARNT protein was in vitro translated from pcDNA3-ARNT, which was provided by Dr. B. Keith (University of Pennsylvania).

Cells were lysed in buffer containing 6 M guanidinium–HCl and protein–ubiquitin conjugates were captured by immobilized metal affinity chromatography (IMAC) on Nickel-Agarose beads (Qiagen) and washed in 8 M urea. After release from the beads conjugates were resolved by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected by immunoblotting. The antibodies used are listed in Supplementary Experimental Procedures.
In vitro ubiquitination assays were performed as described elsewhere (33 (link)). Briefly, ³⁵S-labelled ELK-1 was generated by cell-free expression using the coupled TNT reticulocyte lysate system (Promega). After removal of unincorporated ³⁵S-methionine by gel filtration (Micro-Spin, BioRad), radio-labelled ELK-1 was incubated with UBQ (10 μg), rE1 (500 ng), rE2 (UBCH5; 500 ng), ATP (4 mM), DTT (1 mM), ubiquitin aldehyde (5 μM) and HeLa Nxt (15–30 μg) for 1 h at 30°C. Reactions were resolved on 8% SDS-PAGE gels, dried and visualized by phosphor-imaging (Fujifilm).

³⁵S-Methionine-labeled SENP2-C-CS proteins were synthesized by the TNT reticulocyte lysate system (Promega, Madison, WI, USA). Two micrograms of recombinant GST, GST-Smad4-MH1, and GST-Smad4-Linker proteins prepared from bacteria was subjected to electrophoresis on 12% SDS-PAGE in duplicate. One copy was subjected to Coomassie blue staining for the protein loading control, and the other one was transferred to a nitrocellulose membrane. The membrane was blocked with PBST (phosphate-buffered saline and 0.1% Tween 20) which contained 5% milk for 2 h, and then incubated with isotope-labeled SENP2-C-CS for 4 h. After being washed with PBST three times, the sample was analyzed by autoradiography.