Tran35s label

S-30 extracts were prepared from S. aureus JE2 by cryomilling cell disruption (see “Ribosome profile analysis” below). A runoff reaction was performed by incubating the lysate at 25°C for 70 min with 0.15 volume of runoff premix (0.75 M HEPES [pH 7.5], 7.5 mM dithiothreitol [DTT], 21.3 mM magnesium acetate, 75 μM twenty l-amino acids, 6 mM ATP, 20 mg/ml phosphoenolpyruvate, 50 U pyruvate kinase) relative to the volume of lysate input. The extracts were then dialyzed in Slide-A-Lyzer cassettes (Thermo Fisher) against three changes of buffer A (20 mM HEPES [pH 7.5], 14 mM magnesium acetate, 100 mM potassium acetate, 1 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride [PMSF]), centrifuged at 4°C at 20,800 × g for 10 min, and stored at −80°C.
Linear DNA fragments containing the hpf promoter fused to a gfp or a luc reporter were PCR amplified with P630/P631 (Table 1) using pLI50gfp or pLI50luc as a template. Typical 25-μl reaction mixtures contained 500 ng of DNA template, 10 μl of translation premix (81 (link)), 2.5 μl of 1 mM l-amino acids lacking methionine, 7.5 μl of S-30 extract, 200 ng/μl anti-ssrA oligonucleotide (5′-TTAAGCTGCTAAAGCGTAGTTTTCGTCGTTTGCGAGTA-3′), and 10 μCi Tran³⁵S-label (MP Biomedicals). After a 1-h incubation at 37°C, protein samples were precipitated in 4 volumes of acetone, resolved on 4% to 20% TGX SDS-PAGE gels (Bio-Rad), and autoradiographed.

Tran³⁵S-Label (MP Biomedicals, Illkirch, France) was added to the culture medium (0.37 MBq/ml) for the final 2 h of culture. Cells were lysed in RIPA buffer with protease inhibitors. Lysates were supplemented with 10 mg/ml L-cysteine and 10 mg/ml L-methionine, clarified by centrifugation, applied onto Whatmann filter discs (GE Healthcare, UK), and air dried. Bound proteins were precipitated by 10% (w/v) trichloroacetic acid (Sigma, UK). Boiling 5% (w/v) trichloroacetic acid was added to the filter discs before washing in absolute ethanol and acetone. The filter discs were air-dried, immersed in OptiScint ‘HiSafe’ scintillation fluid (PerkinElmer, UK) and radioactivity was measured using a WALLAC 1409 liquid scintillation counter (PerkinElmer, USA). Control cells were treated with cycloheximide (10 μg/ml). Counts from cycloheximide-treated samples were subtracted from experimental values and the difference was expressed as a percentage of untreated cells.

For GST pull-down analyses, equal amounts of GST, GST-MDM2 and GST-MDM2 deletion mutants, respectively, were immobilized on Gluthation-Sepharose beads (# 17-0756-01, GE-Healthcare), washed five times with GST low salt buffer (50 mM Tris–HCl, 200 mM NaCl, 0.8 mM EDTA, 0.1% NP40, 1 mM PMSF, 10 µg/ml aprotinin) and incubated with equal amounts of NIR full-length protein or NIR deletion mutants (in vitro translated from pCMX-myc-NIR full length or pCMX-myc-NIR (3–245), (147–609), (609–749). The in vitro translation was performed with the TNT-T7 Coupled Reticulocyte Lysate System (# L4610), according to the manufacturer’s protocol (Promega), with 1 µg of plasmid and ³⁵S radiolabelled cysteine and methionine (Tran³⁵S-Label™, # 51006, MP Biomedicals). After overnight incubation at 4°C, all probes were washed five times with GST high salt buffer (50 mM Tris–HCl, 500 mM NaCl, 0.8 mM EDTA, 0.1% NP40). GST protein complexes were eluted from the sepharose by adding SDS-sample buffer [100 mM Tris–HCl (pH 6.8), 100 mM DTT, 4% SDS and 20% glycerol] and by boiling samples for 10 min. The proteins were separated by SDS-PAGE, immobilized on PVDF membrane (Immobilon P, #IPVH00010, Millipore) and visualized by autoradiography. GST-proteins were detected with the monoclonal anti-glutathione-s-transferase antibody (clone GST-2).

The rOsCaM1 protein [116 (link)] was prepared according to Fromm and Chua [117 (link)] to be used as a probe for cDNA library expression screening. Production of the recombinant protein in 50 ml cell culture of BL21(DE3) cells harboring pET-21a(+) containing a complete OsCam1–1 ORF was induced by IPTG. After 15 min of IPTG induction, 1 mCi or Tran ³⁵S Label™ (MP Biomedicals, USA) was added to the culture, and the incubation was continued for 4 h. After centrifugation, the pelleted cells were resuspended in 4 ml B-PER reagent containing 1 U/ml DNase I and 20 μg/ml lysozyme. The homogenate was heated in boiling water for 5 min and centrifuged at 15,000 xg for 10 min. The supernatant was collected, and the ³⁵S-labeled rOsCaM1 protein was purified by phenyl-sepharose chromatography in the presence of Ca²⁺ according to Liao and Zielinski [118 ].

Cells were seeded at 3 × 10⁵ viable cells/ml in spent media and wells were spiked with 762 kBq of ³⁵S radiolabel (TRAN³⁵S‐LABEL™; MP Biomedicals, LLC) and incubated at 36.5°C with 5% CO₂ for 20 h. Following incubation, wells were harvested, and acetone precipitation of the supernatant was performed, and the protein pellet resuspended in radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM HEPES, with a protease inhibitor cocktail tablet; Roche). Cell pellets were either lysed in RIPA buffer or used for a pulse‐chase experiment. The pellet for pulse‐chase was resuspended in fresh media incubated at 36.5°C with 5% CO₂ for a further 24 h following which the wells were harvested in the same manner as before. Samples could then be analyzed by SDS‐PAGE with further preparation by immunoprecipitation if required. Following Coomassie staining, dried gels were then exposed to ECL hyper film (GE Healthcare).