G 25 column

Metabolic labeling of nascent proteins with OP-puro was performed as previously described (Iwasaki et al., 2019 (link)). Cells were treated with 20 µM OP-puro and incubated at 37°C for 30 min in a CO₂ incubator. After washing with PBS, cells were lysed with buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, and 1% Triton X-100. Nascent polypeptides were labeled with IRdye800CW Azide (LI-COR Biosciences) with a Click-iT Cell Reaction Buffer Kit (Thermo Fisher Scientific). After free dye was removed by a G-25 column (Cytiva), labeled polypeptides were separated by SDS-PAGE. The gel was imaged by Odyssey CLx (LI-COR Biosciences) for the detection of nascent peptides with infrared at 800 nm. Then, total proteins were stained with CBB (FUJIFILM Wako Chemicals) and imaged with an infrared 700 nm signal. The gel area ranging from 17 kDa to 280 kDa was quantified using Image Studio (version 5.2, LI-COR Biosciences), and the nascent peptide signal was normalized to the total protein signal.

Total RNA was extracted from cells by TRIzol Reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. Purified RNAs were electrophoresed on Super Sep RNA gels (FUJIFILM Wako Chemicals), transferred onto nylon membranes (Biodyne, Thermo Fisher Scientific), and then UV-crosslinked. The membranes were incubated with UltraHyb-Oligo (Thermo Fisher Scientific) at 37°C for 1 hr. DNA oligonucleotide probes (see below for details) were radiolabeled with [γ−³²P] ATP (PerkinElmer) by T4 PNK (New England Biolabs) and purified with a G-25 column (Cytiva). After prehybridization, membranes were incubated with a labeled DNA probe overnight at 37°C and washed three times with 2× saline-sodium citrate solution. The signal on the membranes was detected by an Amersham Typhoon (Cytiva) scanner. DNA oligonucleotide sequences used as probes are listed below:
tRNA^Tyr_GUA: 5′-ACAGTCCTCCGCTCTACCAGCTGA-3′, tRNA^Leu_HAG: 5′-CAGCGCCTTAGACCGCTCGGCCA-3′, and U6: 5′-CACGAATTTGCGTGTCATCCTT-3′.

The 5′-end labeling of RNA was performed using 5 pmol of RNA, 1 μL γ-³²P-ATP (PerkinElmer), and 10 U T4 polynucleotide kinase (New England Biolabs) in a final volume of 10 µL. Before labeling, RNA was boiled for 3 min and snap-cooled by placing on ice for another 3 min. The radiolabeled RNA was purified on a G-25 column (Cytiva) according to the manufacturer’s instructions. The radiolabeled RNA concentration was determined based on a standard curve which was obtained from the counts per minute of the γ-³²P-ATP source.

Small unilamellar liposomes were
prepared by thin-film hydration and extrusion. POPC and MPB-PE in
chloroform (Avanti Polar Lipids, Alabaster, Alabama) were mixed to
achieve a ratio of 95:5 mol%. A dried lipid film was obtained by evaporating
chloroform using a nitrogen stream and placing the film in a vacuum
desiccator overnight to completely remove the solvent. The lipid film
was hydrated with 0.01 M PB at pH 7.4 filtrated through a 0.2 μm
filter or 50 mM carboxyfluorescein dissolved in 10 mM PB with 90 mM
NaCl at pH 7.4. The lipid suspension was then placed on a shaking
table for 10 min and vortexed for 1 min, resulting in a lipid concentration
of 5 mg/mL. Monodisperse vesicles were obtained by extruding the suspensions
21 times through a 100 nm polycarbonate membrane using a mini extruder
(Avanti Polar Lipids, Alabaster, Alabama). Prior to further analysis,
unencapsulated CF was removed from lipid suspensions hydrated in CF-stock
by size exclusion chromatography on a G-25 column (Cytiva, Marlborough,
Massachusetts) in PBS.

The 5′-end labeling of RNA was performed using 5 pmol of RNA, 1 μL γ–³²P-ATP (PerkinElmer) and 10 U T4 polynucleotide kinase (New England Biolabs) in a final volume of 10 μL. Before labeling, RNA was boiled for 3 minutes, and snap cooled by placing on ice for another 3 minutes. The radiolabeled RNA was purified on a G-25 column (Cytiva) according to the manufacturer’s instructions. The radiolabeled RNA concentration was determined based on a standard curve which was obtained from the counts per minute of the γ–³²P-ATP source.

Protein masses were determined on purified solution samples. To this end, the proteins in 8 M urea containing buffer were loaded onto a G-25 column (Cytiva, Marlborough, MA, USA) and eluted with 10 mM ammonium acetate. After a concentration step using Zip Tip C4 (Merck Millipore Darmstadt, Germany), 1 μL of protein at ~1.5 mg/mL was mixed with 1 μL of sinapinic acid matrix solution in 0.3% TFA/CH₃CN (50:50 v/v). One μL of the mix was spotted on the target and analyzed by MALDI-TOF on a Ultraflex III spectrometer (Bruker Daltonics, Wissembourg, France) controlled by the Flexcontrol 3.0 package (Build 51) and operated in the linear mode, using a maximum accelerating potential of 25 kV and a 20,000–100,000 m/z range (LP_66kDa method). The laser frequency was fixed to 100 Hz and ~1000 shots per sample were cumulated. Four external standards (Protein Calibration Standard II, Bruker Daltonics) were used to calibrate each spectrum to a mass accuracy within 100 ppm. Peak picking was performed using the FlexAnalysis 3.0 software with an adapted analysis method. Parameters used were the centroid peak detection algorithm, S/N threshold fixed to 5 and a quality factor threshold of 30.