Complete protease inhibitor cocktail

Whole‐cell lysates of Calu‐3 cells were prepared using a Triton‐X lysis buffer containing “cOmplete protease inhibitor cocktail” (Roche). Protein concentrations were assessed via the “DC Protein assay” (Bio‐Rad Laboratories). Proteins were separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (ThermoFisher Scientific). For protein detection the following primary antibodies were used: GAPDH (1:1000, R&D Systems, #2275‐PC‐020), ISG15 (1:200, Santa Cruz, #sc166755), MX1 (1:1000, Cell Signaling, #37849), pSTAT1 Y701 (1:1000, Cell Signaling, #9167), pSTAT2 Y689 (1:1000, Upstate, #02‐224), SARS‐CoV‐2 Nucleoprotein (1:1000, Sino Biological, #40143‐R019), STAT1 (1:1000, Cell Signaling, #14994), STAT2 (1:1000, Upstate, #06‐502) and Tubulin (1:1.000, Abcam, #ab179513). Protein bands were visualized using IRDye‐labeled secondary antibodies at dilution 1:40 000 (LI‐COR Biotechnology, IRDye 800CW Goat anti‐Rabbit, #926‐32211 and IRDye 800CW Goat anti‐Mouse IgG, #926‐32210) and Odyssey Infrared Imaging System (LI‐COR Biosciences).

Approximately 1 × 10¹⁰T. brucei 29-13 cells were harvested and washed with PBS, and mitochondria were enriched as described previously (Hashimi et al. 2008 (link); McAdams et al. 2018 (link)). Enriched mitochondria were lysed in N150 buffer (50 mM Tris [pH 8], 150 mM NaCl, 0.1% [v/v] NP-40, and 5 mM β-ME) with 1% (v/v) Triton X-100 in the presence of cOmplete Protease Inhibitor Cocktail (Roche). Lysate was divided into two fractions: one was incubated with 200 U SUPERase-In RNase Inhibitor (Invitrogen) and 0.5 μg/mL DNase I, while the other was incubated with DNase I and a nuclease cocktail containing 60 μg RNase A, 2500 U RNase T1 (Ambion), 28 U RNase H (Invitrogen), and 2040 U micrococcal nuclease for 1 h on ice. Lysates were then incubated with α-DRBD18 antibodies (Lott et al. 2015 (link); Ciganda et al. 2023 (link)) attached to protein A fast-flow beads (GE Healthcare) for 2 h at 4°C. After washing with wash buffer (50 mM Tris [pH 8], 300 mM NaCl, 0.1% [v/v] NP-40, and 5 mM β-ME), the bound proteins were electrophoresed on a 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) and probed with polyclonal antibodies against DRBD18 (Lott et al. 2015 (link)), RESC2 (Ammerman et al. 2011 (link)), RESC8 (McAdams et al. 2019 (link)), RESC10 (Dubey et al. 2021 (link)), RESC11A (Simpson et al. 2017 (link)), RESC13 (Fisk et al. 2008 (link)), RESC14 (McAdams et al. 2018 (link)), KRBP72 (Dubey et al. 2024 ), KH2F2 (Kumar et al. 2016 (link)), MRP1 (Fisk et al. 2009a (link)), MRP2 (Fisk et al. 2009a (link)), P22 (Hayman et al. 2001 (link)), RBP16 (Ammerman et al. 2008 (link)), TbRGG1 (Fisk et al. 2009a (link)), and RECC complex (KREPA1, KREPA2, KREPA3, and KREL1) (Panigrahi et al. 2001 (link)). Blots were washed with TBST buffer and subsequently probed with secondary antibodies, either goat α-rabbit HRP (1:2000 dilution) or goat α-mouse HRP (1:2000). Signals were detected using an ECL preparation as recommended by the manufacturer (Thermo Fisher Scientific), visualized on a Chemi Doc imaging system (Bio-Rad).
For pulldown in the reverse direction, 1 × 10¹⁰ cells harboring endogenously tagged RESC13-MHT, RESC14-MHT, or RESC2-PTP (Wackowski et al. 2024 (link)) were harvested and lysed in N150 buffer with 1% (v/v) Triton X-100 in the presence of cOmplete Protease Inhibitor Cocktail (Roche), 1 mM CaCl₂, and 0.5 μg/mL DNase I. Lysate was then divided into two fractions: one fraction was incubated with 200 U SUPERase-In RNase Inhibitor (Invitrogen) and 0.5 μg/mL DNase I, whereas the other fraction was incubated with DNase I and a nuclease cocktail containing 60 μg RNase A, 2500 U RNase T1 (Ambion), 28 U RNase H (Invitrogen), and 2040 U micrococcal nuclease for 1 h on ice. The two lysates were then incubated with IgG Sepharose 6 Fast Flow beads (Cytiva) for 2 h on ice. Beads were washed with N150 buffer, and for MHT-RESC13 and MHT-RESC2, they were incubated in TEV cleavage buffer (10 mM Tris [pH 8.0], 150 mM NaCl, 0.1% [v/v] NP-40, 0.5 mM EDTA, 1 mM DTT) with 100 U AcTEV Protease (Invitrogen) at 4°C overnight. For MHT-RESC14, glycine (100 mM [pH 3.0]) elution, followed by neutralization with 1 mM Tris-HCl (pH 8.0), was performed. IP was performed as described previously (McAdams et al. 2018 (link); Wackowski et al. 2024 (link)). TEV elutions (RESC13 and RESC2) and glycine elution (RESC14) were subjected to western blot analysis to detect the target protein using either native antibodies (RESC2 and RESC13) or α-Myc antibodies (Invitrogen; 1:2000 dilution) (RESC14), and interacting partner DRBD18 was detected using α-DRBD18 antibody.
For pulldown of DRBD18 overexpression variants, 1 × 10¹⁰ cells were harvested, and then washed in 1× PBS. Cell lysates were precleared using Superdex 200 beads (Cytiva). IP of 2XMyc-tagged DRBD18 variants from cleared lysate was performed using α-Myc antibody as described previously (McAdams et al. 2018 (link)). Glycine elutions were subjected to western blot analysis to detect the DRBD18 variants using α-Myc antibody and interacting RESC factors (RESC11A, RESC2, and RESC13) using their native antibodies.

In general, Flp-In T-REx 293 cells were co-transfected with 1 µg plasmid per construct in a 6-well plate using TransIT-293 according to the manufacturer’s instructions. 16–24 hr after transfection, the cells were collected in cold PBS, lysed in lysis buffer (50 mM HEPES pH 7.5, 100 mM KOAc, 2.5 Mg(OAc)₂, 1% digitonin with 1 mM dithiothreitol [DTT], 1 × cOmplete protease inhibitor cocktail [PIC; Roche, 1187358] with or without 1 × PhosStop (Roche)) for 10 min on ice, and clarified by centrifugation at 21,130 × g for 10 min at 4 °C. Lysis buffer for Strep-Tactin pulldowns (PD) also contained BioLock (IBA, 2–0205). Normalized lysates were added to immunoprecipitation (IP) buffer (50 mM HEPES pH 7.5, 100 mM KOAc, 2.5 Mg(OAc)₂, 1% Triton X-100) containing either anti-FLAG M2 agarose (Sigma, A2220) or Strep-Tactin Sepharose HP resin (Cytiva, 28-9355-99) and incubated while rotating for 1 hr at 4 °C. After rotation, samples were washed 3 times with IP buffer and eluted in protein sample buffer.
To immunoprecipitate endogenously tagged CDAN1 or CDIN1, 12 15 cm dishes of each cell line were lysed in 12 mL IP buffer supplemented with 1 mM DTT, 1 × PIC, and 1 × PhosStop and incubated with anti-FLAG M2 agarose resin. Beads were washed, eluted in protein sample buffer, and analyzed using SDS-PAGE and Coomassie staining.

For protein extraction, 30 mg of brain homogenate tissue was lysed in ice-cold RIPA buffer (50 mM Tris-HCl, pH 8; 150 mM sodium chloride; 1 mM EDTA; 1% Triton X-100; 0.5% sodium deoxycholate; 0.1% sodium dodecyl sulfate) supplemented with complete protease inhibitor cocktail (Roche, Mannheim, Germany). The final protein concentration in the supernatants was determined using a Pierce BCA assay kit (Thermo Fisher, Waltham, MA, USA), and a total of 25 µg of protein was subjected to 4 − 15% Mini-PROTEAN polyacrylamide gel electrophoresis (Bio-Rad Laboratories, Hercules, CA, USA) and transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad Laboratories) using a mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad Laboratories). The membranes were then blocked for 1 h at room temperature with Tris-buffered saline containing 5% nonfat dry milk and 0.1% Tween-20 and incubated with anti-caspase1 (1:1,000; clone EPR16883, Abcam, Cambridge, UK, ab179515) and anti-beta-tubulin (1:2,500; clone AA2, MerckMillipore, Burglinton, MA, USA, T5076) antibodies overnight at 4 °C. The membranes were finally incubated with an enhanced chemiluminescence (ECL) horseradish peroxidase-conjugated secondary antibody (1:5,000, Bethyl Laboratories, Montgomery, TX, USA, A90-216P) for 1 h at room temperature. Images were obtained by using the Octoplus QPLEX imaging system after the membranes were incubated with Clarity Max ECL substrate reagent (Bio-Rad Laboratories).

The construction of pEAQ-HT-PV3 SktSC8-P1 and pEAQ-3CD, containing plant codon-optimised versions of the P1 region of PV3(Saukett)-SC8 and the PV 3CD protease from PV1 Mah have been reported previously^{38 (link)}. Plasmids pEAQ-HT-PV1 MahSC6b-P1 and pEAQ-HT-PV2 MEFSC6b-P1 were constructed by inserting codon-optimised versions of the P1 region of either PV1 (Mahoney)-SC6b or PV2 (MEF-1)-SC6b between the AgeI and XhoI sites of pEAQ-HT^{66 (link)}. Agrobacterium tumefaciens strain LBA4404 was transformed with each construct separately by electroporation and propagated at 28 °C in Luria-Bertani media containing 50 µg/mL kanamycin and 50 µg/mL rifampicin. Co-expression of the P1 sequences with the 3CD protease in Nicotiana benthamiana leaves was carried out by co-infiltration as previously described^{38 (link)} (Supplementary Fig. 4).
At various days post-infiltration (dpi) VLPs were extracted from the infiltrated region of the leaves with Dulbecco’s phosphate-buffered saline + MgCl₂ and CaCl₂ (Sigma-Aldrich), in each case supplemented with EDTA to a final concentration of 20 mM. All extraction buffers also contained cOmplete protease inhibitor cocktail (Roche, UK). The purification process was carried out as previously described^{38 (link)}. Briefly, crude extracts were centrifuged at 9500 × g for 15 min at 4 °C following filtration over a 0.45 µm syringe filter (Sartorius). The clarified extract was then concentrated through a sucrose cushion (1 ml 70% (w/v) and 5 ml 25% (w/v)) at 167,000 × g for 3 h at 4 °C and the lower fraction retrieved. Following dialysis and further concentration using PD10 desalting columns (GE Healthcare) and Amicon Centrifugal Filter Units (Millipore), the sample was purified by ultracentrifugation through a Nycodenz (Axis-Shield) gradient (20–60% (w/v)) at 247,103 × g for 24 h and 4 °C. VLPs were collected by piercing the side of the tube with a needle and the Nycodenz removed through PD10 desalting columns (GE Healthcare) prior to concentration using Amicon Centrifugal Filter Units (Millipore) (Table 1 and Supplementary Fig. 4).