Protein immunoprecipitation and immunoblotting

Equal amounts of protein from cell lysates were subjected to SDS-PAGE and transferred to a PVDF membrane (Millipore). HeLa GFP and HeLa TRAP1-GFP cells were treated with either 2 μg/mL of harringtonine and 100 μg/mL of emetine for 15 min at 37°C to inhibit cytosolic translation or 30 μM linezolide or 200 ng/mL chloramphenicol for 1 hr at 37°C to inhibit mitochondrial translation. eGFP-fusion proteins were immunoprecipitated with GFP-trap magnetic agarose beads (GFP-trap_MA Chromotek) according to manufacturer’s instructions. The following antibodies were used for WB, immunofluorescence and immunoprecipitation: anti-TRAP1 (Santa Cruz Biotechnology, sc-13557 and Genetex, GTX102017), anti-β-ACTIN (Santa Cruz Biotechnology, sc-69879), anti-puromycin (Merck, MABE343), anti-HSP90 (Santa Cruz Biotechnology, sc-1057), anti-Tubulin (Sigma-Aldrich, T9026), anti-TOM40 (Genetex, GTX133780), anti-TIM44 (Santa Cruz Biotechnology, sc-390755), anti-TIM23 (Santa Cruz Biotechnology, sc-514463), anti-TOM20 (Santa Cruz Biotechnology, sc-17764), anti-GAPDH (Santa Cruz Biotechnology, sc-69778), anti-PHB2 (Santa Cruz Biotechnology, sc-133094), anti-TUFM (Genetex, GTX101763), anti-eEF1a (Millipore, #05-235), anti-IDH2 (Genetex, GTX133078), anti-Rieske (Santa Cruz Biotechnology, sc-271609), anti-STAT1 (Santa Cruz Biotechnology, sc-346), anti-RPS6 (Santa Cruz Biotechnology, sc-74459), anti-RPL19 (Santa Cruz Biotechnology, sc-100830), anti-MRPS5 (Genetex, GTX103930), anti-GFP (Santa Cruz Biotechnology, sc-81045). Total protein normalization has been performed by using No-Stain^™ Protein Labeling Reagent (ThermoFisher Scientific, Cat.n. A44717). Images were acquired with a Chemidoc MP imaging system (Bio-Rad), and, where indicated, protein levels were quantified by densitometric analysis using the software ImageJ (25 (link)).

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Avolio R., Agliarulo I., Criscuolo D., Sarnataro D., Auriemma M., Pennacchio S., Calice G., Ng M.Y., Giorgi C., Pinton P., Cooperman B., Landriscina M., Esposito F, & Matassa D.S. (2023). Cytosolic and mitochondrial translation elongation are coordinated through the molecular chaperone TRAP1 for the synthesis and import of mitochondrial proteins. bioRxiv.

Publication Preprint 2023

Actin Agarose Antibodies Cell Chloramphenicol Cytosolic Densitometric Egfp proteins Emetine Gapdh Harringtonine Hela cells Hsp90 Idh2 Immunofluorescence Immunoprecipitation Inhibit Linezolide Membrane Mitochondrial Phb2 Protein Puromycin Pvdf Sds page Stain Stat1 Trap1 Tubulin

Corresponding Organization : University of Naples Federico II

Other organizations : Institute for Experimental Endocrinology and Oncology, National Research Council, Centro di Riferimento Oncologico della Basilicata, Istituti di Ricovero e Cura a Carattere Scientifico, University of Pennsylvania, University of Ferrara, University of Foggia

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Variable analysis

independent variables

Inhibition of cytosolic translation with 2 μg/mL of harringtonine and 100 μg/mL of emetine for 15 min at 37°C
Inhibition of mitochondrial translation with 30 μM linezolide or 200 ng/mL chloramphenicol for 1 hr at 37°C

dependent variables

Protein levels of TRAP1-GFP fusion protein
Protein levels of various mitochondrial and cytosolic proteins (e.g., β-ACTIN, HSP90, TOM40, TIM44, TIM23, TOM20, GAPDH, PHB2, TUFM, eEF1a, IDH2, Rieske, STAT1, RPS6, RPL19, MRPS5)

control variables

Equal amounts of protein from cell lysates
HeLa GFP and HeLa TRAP1-GFP cell lines
Immunoprecipitation of eGFP-fusion proteins using GFP-trap magnetic agarose beads
Total protein normalization using No-Stain™ Protein Labeling Reagent

controls

Positive control: HeLa GFP cell line
Negative control: Not explicitly mentioned

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