Pro mix

For general protein synthesis, 2 × 10⁵ K562C or PC3 cells were incubated for 30 min with [³⁵S] methionine/cysteine (PRO-MIX, GE Healthcare, >1000 Ci/mmol) to a final concentration of 10 μCi/ml. Cells were lysed in PBS–SDS buffer (150 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.4 mM KH₂PO₄ and 0.1% SDS) and proteins were precipitated in 10% (w/v) trichloroacetic acid (TCA). After three washes with 5% (w/v) cold TCA, the insoluble material was collected on GFC filters (Whatman) and the incorporated radioactivity was measured in scintillation counting.
For immunoprecipitation, PC3 cells (4 × 10⁶) or K562C (15 × 10⁶) were incubated in 2.5 or 5 ml of methionine- and cysteine-free medium supplemented with 10% dialyzed FBS and containing [³⁵S]methionine/cysteine (PRO-MIX, GE Healthcare, >1000 Ci/mmol) to a final concentration of 300 μCi/ml for 30 min at 37°C. The labeling medium was removed, and the cells were washed once in PBS, frozen in liquid nitrogen and stored at −70°C to be analyzed later or immediately processed.

For general protein synthesis analysis, 1 × 10⁶ lymphoblastoid cells were incubated for 30 min with [³⁵S]methionine/cysteine (PRO-MIX, GE Healthcare, > 1000 Ci/mmol) to a final concentration of 10 μCi/ml. Cells were lysed in PBS–SDS buffer (150 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.4 mM KH₂PO₄ and 0.1% SDS) and proteins were precipitated in 10% (w/v) trichloroacetic acid (TCA). After three washes with 5% (w/v) cold TCA, the insoluble material was collected on GFC filters (Whatman) and the incorporated radioactivity was measured in scintillation counting.

Immunoprecipitation experiments were performed in cells at the exponential phase grown in TAP medium under continuous light at 70 μmol photons m^–2 sec^–1, as described by De Marchis et al. (2018 (link)) for Nicotiana tabacum (tobacco) protoplasts, with minor modifications. In brief, approximately 3 million algae cells were subjected to pulse labelling for up to 2 h by using Pro‐Mix – a mixture of [³⁵S]Met and [³⁵S]Cys (GE Healthcare, https://www.gehealthcare.com). After the pulse, the chase was performed by adding unlabeled Met and Cys to final concentrations of 10 and 5 mm, respectively. Cells were sampled at different pulse and chase time points. The cells were homogenized by adding homogenization buffer (150 mm Tris‐Cl, pH 7.5, 150 mm NaCl, 1.5 mm EDTA, 2% Triton X‐100 and complete protease inhibitor cocktail; Roche, https://www.roche.com) to frozen samples. Proteins were immunoselected using rabbit polyclonal antisera against D2. The immunoprecipitates were analyzed using SDS‐PAGE. After electrophoresis, gels were treated with Amplify™ fluorography reagent (GE Healthcare), dried and exposed for fluorography.