Nuclease treated rabbit reticulocyte lysate

In vitro translation assay was performed as described in [ref. ^{40 (link)}]. Briefly, translation of luciferase mRNA (Promega) was performed with nuclease-treated rabbit reticulocyte lysate (Promega). Each reaction mix (30 µl) contained the following components: 12.6 µl of rabbit reticulocyte lysate, 0.5 µl of Luciferase mRNA (1 mg/ml), 0.3 μl of amino acid mixture minus leucine (1 mM), 0.3 μl of amino acid mixture minus methionine (1 mM), and 16.3 μl of proteins, puromycin (Sigma) or buffer (25 mM NaPO4 pH 7.4, 50 mM NaCl, 2 mM DTT). Reactions were assembled at different concentration of RGG or RGG-Ub (20-10-5-2.5-1.2-0.6-0.3 µM) or BSA (10 µM). All in vitro translation reactions were prepared on ice, followed by incubation at room temperature for 3 h before luminescence measurement. Luciferase translation was measured with luciferase assay system (Promega) by mixing 75 µl of luciferase substrate with 3 µl of unpurified translation mixture in a black 96-well plate (Corning). End-point luminescence measurements were carried out in triplicate using Victor3 multilabel plate reader (Perkin Elmer).

Endofin(1-84) constructs encoded on a pTriex5 vector modified to generate a C-terminal Strep-tag were amplified using Pwo Polymerase (Roche). RNA was synthesized from the PCR product using T7 RNA polymerase (Promega) and protein was translated in nuclease treated rabbit reticulocyte lysate (Promega) containing ³⁵S-methionine (Perkin Elmer), and 100 units of RNasin (Promega) for 1h at 30°C. Samples were incubated at 30°C with 1 mM puromycin for a further 10 min. For binding experiments, 20 μl translated protein was incubated with 5 μg His₆-HD-PTP_Bro1-CC in 250 μl IP2 buffer (20mM Hepes, pH7.4, 100mM NaCl, 1mM MgCl₂, 1% (w/v) Triton X-100) for 2h at 4°C, then overnight with 3 μl anti-His antibody. Samples were incubated with 20μl protein A-sepharose beads (Invitrogen) for 2h at 4°C, then washed 3 x in IP2 buffer. Following SDS-PAGE gels were dried and visualized using phosphorimaging and quantified using AIDA (Raytest). For CHMP4B binding experiments, full length or truncated GST-CHMP4B (50 μg/ml) and His₆-HD-PTP_Bro1-CC (5 μg) were incubated in 200 μl IP2 buffer containing 250 mM NaCl for 2h at 4°C prior to immunoprecipitation with anti-His. For competition experiments endofin 22-mer peptide was added at a final concentration of 20 μM. At least 3 independent replicates were performed for all experiments.

Nuclease-treated rabbit reticulocyte lysate (Promega) was used to perform the cell-free translation. 150 ng/μL (final concentration) of the in vitro transcribed 5′-capped mRNA containing the full-length 5′ UTR attached to the firefly luciferase coding sequence was translated according to the manufacturer’s protocol. In order to measure the firefly luciferase activity, 5 μL of the translation reaction was added to 75 μL of luciferase assay reagent (Promega) at room temperature. The reaction was quickly mixed and luminescence was measured using CLARIO star microplate reader (BMG LABTECH).

Translation occurs in an incubation mixture (30 μl) containing 20 μl of nuclease-treated rabbit reticulocyte lysate (Promega), amino acids (20 μM) and mRNA as indicated in figure legends. Translation was performed at 30°C for the indicated amount of time. New synthetized proteins were visualized by western-blotting using specific antibodies (anti-luciferase, anti-HA).