Tnt system

GST-fused constructs were produced in BL21 Escherichia coli, and the bacterial lysates were collected using ultrasound. In vitro transcription and translation experiments were performed using rabbit reticulocytes (TNT system, Promega, USA) according to the manufacturer’s recommendations. In the GST pull-down assay, ~5 μg of the appropriate GST-fused protein with 25 μL glutathione-Sepharose beads were mixed with 40 μL of the in vitro transcribed/translated products and the protease inhibitor cocktail by constant rotation at 4 °C for 2 h. Beads were washed five times with binding buffer and then resuspended in 25 μL of 2 × SDS-PAGE loading buffer. The fused protein was measured by western blotting.

The DNA of pSP64-HPV16 E6, pSP64-p53-pro and pCDNA3-MAGI-3 plasmids was transcribed and translated in vitro in a rabbit reticulocyte lysate (Promega TNT System, Madison, Wisconsin, USA), following the manufacturer instructions, with radiolabeling with 0.6 mCi/mL ³⁵[S]-cysteine (GE Healthcare). The translation efficiency was monitored by analyzing 1 µL aliquots of each protein using SDS-PAGE and PhosphorImager analysis (Fujifilm, Milano, Italy).
In the standard in vitro degradation assay [36 (link)], degradation was monitored by mixing the translated target proteins with 50 ng of the purified His₆-E6 protein at a 3:1 ratio with an incubation at 25 °C. After 1 h and 2 h, 5-µL aliquots were removed from the reaction mixtures and analyzed. All volumes were equalized using the water-primed reticulocyte lysate TNT mix (Promega). The samples were added to 5× loading buffer (250 mM Tris–HCl, pH 6.8, 10 % SDS, 30 % glycerol, 5 % β-mercaptoethanol, 0.02 % bromophenol blue), boiled and analyzed using SDS-PAGE and autoradiography. The degradation experiments were carried out three times, and the relative quantification was performed with a PhosphorImager by measuring the signal intensities of protein bands.

Coupled in vitro transcription-translation reactions were carried out in rabbit reticulocyte lysate supplemented with [³⁵S]methionine using a Promega TNT system according to the manufacturer’s instructions. SDS-PAGE followed by autoradiography was performed according to standard procedures. Immunoprecipitations were performed as previously described (71 (link)). Transfection-based reporter assays to assess host cell shutoff by PA-X (described previously [30 (link)]) were performed by cotransfecting QT-35 cells with a reporter plasmid containing the Renilla luciferase gene along with pHW2000 plasmids expressing the appropriate segment 3 genes with or without the desired PA-X mutations. At 48 h posttransfection, cells were lysed, and luciferase activity was measured on a Promega GloMax 96-well microplate luminometer using the Promega Renilla luciferase system.

A full-length cDNA clone was used to manufacture 35S-radiolabeled 21-hydroxylase antigen with the TNT system (Promega). This was subsequently immunoprecipitated with patient sera. Blood donors served as negative controls and sera from APS-1 patients with known strong reactivity to 21-hydroxylase were used as positive controls. Radioactivity was measured using a liquid scintillation counter (Wallac 1450 MicroBeta, Perkin-Elmer). The cutoff value was set to obtain maximal accuracy, based on the results from a recent inter-laboratory study[17 (link)].

Extraction of the total cell or nuclear lysates and western blot analysis were performed as reported previously [18 (link)]. Reverse transcription PCR using published primer sequences and immunoprecipitation were also performed as described previously [19 (link)]. [³⁵S]Methionine-labeled in vitro translated HIF-1α was prepared using the TNT system (Promega, WI, USA). GST-fusion proteins were expressed in E. coli BL21 (DE3) and induced with 0.4 mM isopropyl-thio-β-D-galactopyranoside. Equal amounts (1 μg) of GST and GST-MTA1 immobilized on glutathione sepharose beads were incubated with in vitro translated HIF-1α in modified GBT buffer (10% glycerol, 50 mM Hepes-NaOH [pH 7.5], 170 mM KCl, 7.5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM DTT, and 1% Triton X-100) at 4°C for 3 h. After washing, the bound proteins were eluted with the sample buffer and were separated by SDS-PAGE, followed by western blot analysis.

E1, E2, Cdh1, and APC/C were expressed and purified as described previously (Carroll et al., 2005 (link); Enquist-Newman et al., 2008 (link)). Substrates were transcribed and translated in vitro using the TNT system (Promega, Madison, WI) from plasmids with ³⁵S-methionine and treated with 10 mM NEM (10 minutes) followed by 20 mM DTT (10 minutes) to inactivate ubiquitin chain-extending activities in the reticulocyte lysate. E1 (Uba1, 300 nM), E2 (Ubc4, 50 µM), ubiquitin (150 µM), and ATP (1 mM) were incubated for 15 minutes. APC/C (0.1–1 nM), substrate (2 µl of TnT mix into 15 µl reaction), and Cdh1 (2 µl of TnT mix into 15 µl reaction) were added. Reaction were incubated for the indicated times at room temperature, stopped by the addition of SDS sample buffer, separated by SDS-PAGE, and visualized and quantified with a Molecular Dynamics Phosphorimager (GE Healthcare, Fairfield, CT).