Imagequant 800

Cells were lysed in RIPA lysis and extraction buffer (Thermo) supplemented with protease inhibitor cocktail and benzonase for 30 min on ice with intermittent vortexing. Protein concentration in total cell lysates was determined using Bradford assay (Bio-Rad) and normalized in all samples before adding 2x SDS sample buffer. Samples were denatured by boiling at 95 °C for 5 min. Proteins were resolved on NuPAGE 4-12% gradient gels (Thermo) with MES or MOPS (Thermo) running buffer at 200 V for 45 min. Proteins were transferred to nitrocellulose or PVDF membranes (Roche) in tris-glycine buffer at 110 V for 1 h. Membranes were washed once in TBS-T and blocked in 5% low-fat dry milk dissolved in TBS-T for 1 h at RT. Subsequently, blots were washed 3 times with TBS-T and probed with primary and secondary antibodies. Chemiluminescence was developed using HRP substrate (Luminata Classico, Merk) and detected on a LAS 4000 (Fuji) or ImageQuant800 (Amersham) imager with control software v1.2.0. AIDA image software v4.27.039 (Elysia Raytest) was used to quantify intensity of protein bands. Images were processed using ImageJ v1.49 s or Adobe Photoshop CC 2018. Uncropped scans of blots in main figures and supplementary figures are provided in Source Data file and at the end of Supplementary Information file, respectively.

Mitochondria and mitoplasts at a concentration of 1 mg/ml were generated by incubating isolated mitochondria from Tim23 WT and Cysteine mutants in SEM (250 mM sucrose, 20 mM MOPS/KOH pH 7.2, 1 mM EDTA) and EM (20 mM MOPS/KOH pH 7.2, 1 mM EDTA) buffer respectively for 15 min on ice. Subsequently, they were treated with Maleimide activated Streptavidin (Thermo Fisher Scientific) at a final concentration of 1 mg/ml and incubated for 30 min on ice followed by 60 min at 25 °C. Samples were harvested by centrifugation and resuspended in protein loading dye containing beta-mercaptoethanol. Following this, they were boiled at 95 °C for 5 min and analyzed by NuPAGE^TM 4−12% Bis-Tris gels and immunoblotting using Tim23^IMS antibody. The images for Tim23^T9C were captured digitally (Amersham™ ImageQuant™ 800) due to the weak detection of Tim23^T9C by this antibody with X-ray films.

Protein was extracted from cells using a lysis buffer (40 mM NaCl, 25 mM Tris pH 8, 2 mM MgCl₂, 0.05% SDS, 2x Complete EDTA-free protease inhibitor (Roche), 0.4 µL mL⁻¹ benzonase) with protein concentration determined by Bradford assay, comparing to BSA standards to ensure loading of an equal mass of protein into each lane. Protein and Laemmli buffer were heated to 100 °C for 5 min before loading into gel. For U2OS, NuPAGE Novex 4–12% Bis-Tris Gel (Invitrogen) was used. For MEFs, NuPAGE Novex 3–8% Tris-Acetate Gel (Invitrogen) was used. Samples were run on gels for 2 hr before transfer from gel to a nitrocellulose membrane. Antibodies used to bind proteins on membrane were pAb rabbit BRIP1/FANCJ antibody (1/10000, Novus Biologicals NBP1-31883), pAb rabbit RTEL1 antibody (1/5000, Novus Biologicals NBP2-22360). For loading control, mAb mouse anti-β-actin antibody (1/5000, Abcam ab8226) was used with U2OS and mAb mouse anti-α-Tubulin (1/10000, Sigma Aldrich T6199) was used with MEFs. Secondary antibodies used were: pAb swine anti-rabbit immunoglobulins/HRP (1/10000, Dako P0217) and pAb goat anti-mouse immunoglobulins/HRP (1/5000, invitrogen A16078). Visualisation was performed by exposure onto photographic film for U2OS and visualisation using Amersham ImageQuant 800 for MEFs. Full uncropped scans are available in the source data file.

1 mg of QA-levan was initially dissolved in 1 mL ultrapure water. Subsequently, pBlueScript II SK (-) plasmid was combined with cationized levan solution to the mass ratio of 0.1 μg levan and 0.2 μg plasmid (1:2), 0.2 μg levan + 0.2 μg plasmid (1:1), 0.3 μg levan + 0.2 μg plasmid (3:2) and 0.4 μg levan + 0.2 μg plasmid (2:1), and stayed at room temperature for 5 min. The formation of QA-levan/DNA polyplexes was analyzed by 1% (w/v) agarose gel electrophoresis in TAE buffer stained with EcoDye™ DNA staining solution (BIOFACT, Daejeon, Republic of Korea). The bands of free DNA and QA-levan/DNA polyplexes in gel electrophoresis was monitored by Amersham ImageQuant™ 800 (Marlborough, MA, USA).

Northern blotting was conducted using NorthernMax-Gly Kit (Invitrogen, AM1946), and a Chemiluminescent Biotin-label Detection Kit (Beyotime, D3308) following manufacturer’s protocols. The circMET junction- and exon-specific probes were listed in Supplementary Table 3. The extracted RNA was separated by agarose gel electrophoresis and transferred to nylon membrane using capillary transfer. After the membrane was fixed by UV, specific probes were added and washed using reagents provided with the kit. Chemiluminescent signals were recorded by Amersham ImageQuant 800 and analysed using Image Lab 6.0.1.

Western blot analysis was performed as previously described protocol (Jeon et al., 2021 (link)). The primary antibodies used were anti-HMGB-1 (Abcam, Cambridge, MA, United States), anti-β-actin (Sigma-Aldrich), anti-KSHV ORF 50 (Bioss, Woburn, MA, United States), anti-HHV ORF 45 (Thermo Fisher Scientific), and anti-KSHV K8.1 antibodies (Santa Cruz, Santa Cruz, CA, United States). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Bethyl; Montgomery, TX, United States) and HRP-conjugated goat anti-rabbit IgG (Bethyl) were used as secondary antibodies. The antibody-reacted membranes were visualized using Amersham ImageQuant 800.

Protein concentrations of whole-cell lysates and membrane fractions were measured using the BCA assay. Cells were resuspended in 20 mM Tris-HCl buffer (pH 8.0) and lysed with lysostaphin (40 μg mL⁻¹) in a 37°C heat block for 30 min. SDS loading buffer at 5× was added to the cell lysates, followed by heating for 5 min. Samples were subjected to 12% SDS-PAGE, and proteins were transferred to polyvinylidene difluoride PVDF membranes (Cytiva). Membranes were blocked in 5% (wt/vol) skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, and 0.05% [vol/vol] Tween 20, pH 7.6) for 1 h. Membranes were washed three times with TBST and incubated with rabbit polyclonal antibodies specific to SaeR (1:1,000 dilution) and SaeS (1:1,000 dilution) in blocking buffer for 1 h (20 (link)). Membranes were washed with TBST and incubated with StarBright Blue 700 goat anti-rabbit IgG (1:3,500; Bio-Rad) for 1 h. After a brief wash in TBST, the signals were visualized using an Amersham ImageQuant 800. The densities (mean intensity per unit area) of the SaeS and SaeR protein bands were determined by quantification with Multi Gauge software (Fujifilm).