Oriole

Luciferase (1 μM) was denatured at 44°C for 10 min in buffer D in the presence of 5 μM sHsps (1.66 μM IbpA_Ec and 3,34 μM IbpB_Ec in case of IbpAB_Ec). To verify sHsps ability to form assemblies, 150 μl of each sample was applied on a 3.6 ml 10–60% glycerol gradient in buffer E (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM Mg acetate, 2 mM DTT). Samples were then centrifuged at 10°C in a Beckman SW 60 rotor at 160,000 g for 1 h, fractions were collected from the top. Protein distribution in each fraction was verified by SDS–PAGE followed by Oriole (Bio-Rad) fluorescent staining.

AAV7m8 GtCCR4-Venus vector was produced by the helper-free triple transfection procedure and purified using affinity chromatography (GE Healthcare)⁴⁰ . Viral titers were determined by quantitative PCR using TaqMan technology (Life Technologies, Gaithersburg, MD, USA). The purity of the vectors was assessed by 4–12% sodium dodecyl sulfate-acrylamide gel electrophoresis and fluorescent staining (Oriole, Bio-Rad, Hercules, CA, USA). The transfer plasmid (pAAV-hSyn-GtCCR4-Venus-WPRE and pAAV-hSyn-ChR2-Venus-WPRE) was constructed by inserting the human synapsin promotor sequence and GtCCR4-Venus fragment with the WPRE sequence into an AAV backbone plasmid, respectively (pAAV-CMV, Stratagene, La Jolla, CA, USA).

Gel electrophoresis was performed using self-prepared 15% polyacrylamide gels or 4–20% ExpressPlus PAGE Gels (Genescript) according to standard procedures and stained with Coomassie brilliant blue or Oriole (Bio-Rad) fluorescent stain. Gel electrophoresis for separation of IbpA_Ec from IbpB_Ec was performed using 15% polyacrylamide gels supplemented with 6M Urea. Immunoblotting was performed according to the standard procedures, using rabbit anti-sera specific for IbpAs and IbpAB as primary antibody, and developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), using (H+L) HRP conjugated anti-rabbit IgG (Bio-Rad) as secondary antibodies. Developed immunoblots were scanned using ChemiDoc MP Imaging System (Bio-Rad) and quantified with ImageLab (Bio-Rad) software.

To evaluate the purity of membranes separated with sucrose density gradient centrifugation described above, OS- or IS-specific marker proteins were quantified in each separated membrane fraction. A marker of OS membranes, visual pigments, was quantified spectrophotometrically as described [4 (link), 9 (link), 10 (link)]. As the markers of membranes in the IS, F1 ATPase β subunit was used for mitochondrial inner membranes, TOM20 for mitochondrial outer membranes, calnexin for endoplasmic reticulum (ER) membranes, and Na⁺/K⁺ ATPase α subunit for IS plasma membranes. The amount of F1 ATPase β subunit was quantified by fluorescent staining with Oriole (Bio-Rad Laboratories, California, USA) after SDS-PAGE using bovine serum albumin (BSA) as a molar standard. The amounts of Na⁺/K⁺ ATPase α subunit and those of calnexin and TOM20 were measured by quantitative immunoblot analysis as described [5 (link)].

Protein in SEC fractions were visualised by SDS-PAGE through staining with Sypro Ruby (Thermo Fisher) or Oriole (Bio-Rad) fluorescent stain, scanned on a Chemidoc (BioRad), and analysed using ImageJ.26 (link),27 (link)
DNA samples were analysed using agarose gel electrophoresis on 1% (w/v) agarose gels in TAE buffer (40 mM Tris, 20 mM acetic acid, and 1 mM EDTA, pH 8.0) containing 0.01% SYBR® Safe DNA Gel Stain (Thermo-Fisher), and visualised using the Chemidoc.
The superoxide dismutase activity was assessed using the in-gel nitro blue tetrazolium chloride (NBT – Sigma-Aldrich, UK) negative staining assay.28 (link) The SEC eluate fractions were resolved on non-denaturing conditions 12% polyacrylamide gels and stained with 0.5 mM NBT, 28 mM TEMED, 28 μM riboflavin in 100 mM sodium phosphate pH 7.0 buffer. The gel was incubated in 20 mL of NBT-riboflavin stain at room temperature in the dark for 20 min, and then visualised by exposing the gel to bright white light. The gels were scanned using a Bio-Rad Chemidoc system with a white light conversion screen.

The PUREfrex^® 2.1 Kit (GeneFrontier, Chiba, Japan) was used as the PURE system reagent for cell-free protein synthesis. For protein synthesis, 5 or 10 µL of the reaction mixture containing 1 (DHFR, sfGFP, and trastuzumab HC) or 2 (Luc and ALP) ng/µL of template DNA was incubated at 23, 30, and 37 °C for the indicated duration, as described in the corresponding figure legends. Following synthesis, the reaction mixture was diluted 50- or 100-fold with water, and the fluorescence of the synthesized sfGFP was measured using a Varioskan™ plate reader (Thermo Scientific, Waltham, MA, USA). For SDS-PAGE analysis, 0.2 or 0.5 µL of the reaction mixture was loaded onto 12.5% or 10–20% gradient gel, and electrophoresis was performed under reducing condition. The gels were stained with Oriole (Bio-Rad, Hercules, CA, USA) or SYPRO Orange Protein Gel Stain (Thermo Scientific). Protein bands were visualized and quantified using ImageQuant Las imager (Cytiva, Tokyo, Japan) or WSE-6300 LuminoGraph III (ATTO, Tokyo, Japan) and CS Analyzer 4 software (ATTO).