Sw41ti

B. subtilis and B. anthracis cells were grown overnight at 37 °C and inoculated to an OD₆₀₀ of 0.05 in 50 ml LB. The LB media was supplemented with 1 mM IPTG for the EF-P complementation strain. Log phase culture (~OD 0.6) was collected by centrifugation at 8000 rpm for 10 minutes (Beckman Coulter Avanti J-15R, rotor JA-10.100). The pellets were resuspended in 200 μl gradient buffer containing 20 mM Tris (pH 7.4 at 4°C), 0.5 mM EDTA, 60 mM NH₄Cl, and 7.5 mM Mg₂ (55 (link)). The cell resuspension was lysed using a homogenizer (Beadbug6, Benchmark) by four 30 s pulses at speed 4350 rpm with chilling on ice for 3 min between the cycles and clarified by ultracentrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24×2). Clarified cell lysates were normalized to 1500 ng/μl and loaded onto 10 – 40% sucrose gradients in gradient buffer. Ultracentrifugation separated ribosome species at 30,000 rpm for 3 hours at 4μC (Beckman Coulter, rotor SW-41Ti). Gradients were collected using a Biocomp Gradient Station (BioComp Instruments) with A260 continuous readout (Triax full spectrum flow cell). The area under each peak for 30S, 50S, and 70S and the several peaks for polysomes, was quantified using Prism (GraphPad).

Polyribosomes were obtained from A. thaliana seedlings and analysed as described previously (42 (link)). Crude polysomal extracts were obtained from 200 mg of Arabidopsis seedlings treated or not with 1 μM auxin during 24 h and resolved on 7–47% sucrose gradient centrifuged for 3 h at 38 000 rpm (rotor SW41-Ti, Beckman Coulter). Analysis of polysome profiles was performed with an absorbance detector at 254 nm and sucrose gradients collected with a BioLogic Duoflow fractions collector (Biorad) into 18 fractions of 500 μl each. Samples from the light (LP), heavy polysome (HP) and non-polysomal 80S, 60S and 40S (NP) fractions were then separated on a 12.5% SDS-PAGE followed by immunoblotting to determine total and phosphorylated RPS6 and RISP protein levels. Rabbit polyclonal antibodies raised against RISP and eL13 were described previously (34 (link)) and (43 (link)), respectively. Polyclonal Phospho-(Ser/Thr)Akt substrate (RxRxxS/T) antibody for RISP-P detection was from Cell Signaling Technology. Antibodies against eS6 (RPS6) and phospho-specific antibodies against S240-P were generated by Agrisera, Umeå, Sweden and kindly provided by Albrecht G. von Arnim (University of Tennessee, Knoxville, USA). Polyclonal anti-eS14 was from Agrisera.

Fibrils were packed into Bruker 3.2 mm NMR rotors via ultracentrifugation at 4 °C and 150,000g for 30 min using Beckman Optima XPN-100 fitted with a SW-41 Ti swinging-bucket rotor and Ultra-Clear tubes. NMR spectra were collected on a 11.75 T magnet (500 MHz 1H NMR frequency) using a Bruker spectrometer and a 3.2 mm Bruker Low-E 1H/13C/15N NMR probe. Two-dimensional 13C-13C NMR spectra were collected using the two-dimensional dipolar assisted rotational resonance NMR technique at 10 kHz magic angle spinning speed with a mixing time of 50 ms. A 1H radiofrequency field of 100 kHz was used. Signal averaging of all the spectra in Fig. 6 required approximately 24 h. This technique produces off-diagonal peaks (crosspeaks) that mostly correspond to interactions between 13C atoms within the same amino acid (64 , 86 , 87 ).

Resistin and RBP4 (Abundant Serum Markers 26-Plex Human ProcartaPlex^™ Panel, USA); omentin (BioVendor, BioCompare), VCAM-1 (Invitrogen^™ eBioscience^™ ProcartaPlex Human VCAM-1 Simplex kit, USA) and isoprostane (8-Isoprostane ELISA Kit, Cayman Chemical Company, USA) will be assessed by enzyme-linked immunosorbent assay (ELISA). Samples will be prepared following the manufacturer’s recommendations and absorbance measured using a microplate reader (Biotek EPOCH 2). Martin’s formula^{21 (link)} will be used to calculate LDL-C during the run-in phase. At randomization and at the end of the trial, lipid profile will be measured by gradient ultracentrifugation.
Continuous density gradient ultracentrifugation (CDGU) will be used to assess very low-density lipoprotein, LDL (subfractions 1–5) and HDL (subfractions 2a, 2b, 3a, 3b and 3c), using a rotor SW41Ti (Beckman Coulter, Brea, CA, USA). TC will be measured in each lipoprotein fraction by enzymatic methods (Cholesterol, Biotechnique, Minas Gerais, Brazil) in the biochemical analyzer SX-140 (Sinnowa, São Paulo, Brazil).
For nitrite and nitrate (NOx) assessment, whole blood will be collected in sodium heparin tube, stored immediately on ice, centrifuged at 3500 rev/min, at 4°C, for 3 min and serum aliquots stored in liquid nitrogen until analysis. Both will be measured by chemiluminescence (model NOA, Sievers Instruments, Boulder, CO, USA).

The sedimentation properties of GTPBP5 and mitoribosomal proteins were analyzed by sucrose gradient sedimentation. From 1.5 to 2 mg of mitochondria isolated from WT or GTPBP5-KO cells were solubilized in lysis buffer containing 260 mM sucrose, 100 mM KCl, 20 mM MgCl₂, 10 mM Tris-HCl, pH 7.5, 1% Triton X100, 1× EDTA-free protease inhibitor cocktail, 0.08 U/μl RNAsin and incubated on ice for 20 min. The lysate was centrifuged at 9200 g for 45 min at 4°C, and the supernatant was loaded onto a 12.5 ml linear 10-30% sucrose gradient containing 10 mM Tris-HCl, pH 7.5, 100 mM KCl, 20 mM MgCl₂ 1× EDTA-free protease inhibitor cocktail, and centrifuged at 24 000 rpm for 15 h at 4°C in SW 41Ti (Beckman Coulter). The gradients were collected from the top into 15 or 16 fractions, TCA (trichloroacetic acid)-precipitated and the pellets resuspended in 1× Laemmli buffer and analyzed by immunoblotting. The continuous RNA profile (absorbance at 254 nm) obtained during gradient collection using a Brandel fractionation system and Brandel PeakChart software version 2.08.
For mass spectrometry analysis, fractions containing monosome, mtLSU, and mtSSU, confirmed by immunoblotting, were pooled and precipitated with methanol/chloroform and analyzed at the Keck Biotechnology Resource Laboratory (Yale University School of Medicine, New Haven, CT, USA).