Brij 58

Flash-frozen rabbit psoas muscles strips with a diameter of 1–2 mm were fully immersed in four 30-min changes of ice-cold fiber preparation buffer (6 mM imidazole, 8 mM Mg-acetate, 70 mM propionate, 5 mM EGTA, 7 mM ATP, and 1 mM Na-azide, pH 7.0). Skinning was then performed by soaking equilibrated samples for 2 h in a fiber preparation buffer with added 0.5% Brij 58 (P5884; Sigma-Aldrich). Skinned samples were stored in the fiber preparation buffer mixed 1:1 with ice-cold glycerol for 2 h before moving to −20°C for long-term storage.
Prior to myofibril preparation, the muscle strips were immersed in a wash buffer (6 mM Mg-acetate, 10 mM EGTA, 54 mM Na-acetate, 18 mM Na₂SO₄, 10 mM MOPS, 5 mM ATP, and 1 mM DTT, pH 7.4). The strips were separated into threads and transferred into a 2-ml tube containing 800 μl of wash buffer. Threads were twice homogenized using a Tissue Ruptor II with a rest period of 30 s (on ice). The myofibril concentration was kept constant by ensuring an OD₆₀₀ of ∼0.8.
No live animals were used in these studies. Muscle tissue was collected in accordance with the U.K. Animals (Scientific Procedures) Act 1986 and associated guidelines.

The process of protein extraction and plasma membrane enrichment were modified from the previously published protocol (Collins et al., 2017 (link)). Briefly, fresh leaf tissues were ground using 1 mL ice-cold buffer H (250 mM sucrose, 50 mM HEPES-KOH pH 7.5, 5% glycerol, 50 mM NaPP, 1 mM NaMo, 25 mM NaF, 10 mM EDTA, 0.5% PVP, 3 mM DTT, 1 mM PMSF) per 0.2 g, and frozen root tissues were ground to a fine powder with 1 mL buffer H per 0.5 g fresh weight. The homogenate was then centrifuged for 10 min (10000 × g, 4°C),and the resulting supernatant was collected and centrifuged in an ultracentrifuge (Thermo Sorvall WX 100) for 30 min (100000 × g, 4°C) to pellet the crude microsomal fractions. The pellets were suspended and incubated in 2 μL of buffer H with 0.02% w/v Brij-58 (Sigma-Aldrich, America) per μg of microsomal protein on ice for 30 min, and then ultracentrifuged for 30 min (100000 × g, 4°C). The Brij-58 treatment and ultracentrifugation were repeated one more time to obtain the final enriched plasma membrane protein fraction.

Pseudovirus (PsV) and Quasivirus (QV) particles were produced by transfecting 293TT cells with HPV16 sheLL plasmids (a gift from the Schiller Lab, NCI, Bethesda, MD, USA) together with either the plasmid for secreted alkaline phosphatase (pSEAP) or cottontail rabbit papillomavirus (CRPV) genome respectively as previously described [23 (link),24 (link),45 (link),46 (link),47 (link)]. Two days post-transfection, cells were collected and lysed using the detergent Brij58 (Sigma-Aldrich, St. Louis, MO, USA). Particles were matured by incubating cell lysates at 37 °C for 24 h and subsequently treating with Benzonase (Sigma-Aldrich, St. Louis, MO, USA) and Plasmid Safe (Epicentre, Madison, WI, USA). Purification of the resulting particles was performed by ultracentrifugation of the lysates at 234,000× g for 3.5 h on an Optiprep step gradient (Sigma-Aldrich, St. Louis, MO, USA). Particles were harvested by puncturing the bottom of the centrifuge tube and collecting fractions of ~250 µL.

Suspensions were prepared from individual colonies after culture on Blood Columbia agar. OD at 600 nm was measured and suspensions were diluted to 10⁹ CFU/ml in EDTA-saline buffer (75 mmol/L NaCl and 25 mmol/L EDTA, pH 7.5), then mixed with an equal volume of 1% low-melting-point agarose and allowed to solidify in a 100 μL plug mould. The agarose plug was incubated for 24 h at 37°C in 500 μL lysis buffer (6 mmol/L Tris–HCl (pH 7.6), 0.1 mol/L EDTA, 1 mol/L NaCl, 0.5% Brij®58 (polyoxyethylene (20) cetyl ether; Sigma), 0.4% sodium deoxycholate, 0.5% sodium lauryl sarcosine and 1 mg/mL lysozyme (and 10 µg/mL lysostaphin to S. aureus)). The lysis buffer was replaced with 500 μL proteinase K buffer (1% sodium lauryl sarcosine, 0.5 mol/L EDTA (pH 9) and proteinase K (50 μg/mL; Sigma)) and this solution was incubated with gentle shaking at 50°C for 20 h. The plugs were then washed four times for 30 min at 37°C with 10 mL of Tris–EDTA buffer (10 mmol/L Tris–HCl (pH 8) and 1 mmol/L EDTA). One-third of a slice of each plug was cut and incubated for 18–20 h with 30 U of SpeI, or SmaI, XbaI and Cfr9I in the restriction buffer (Thermo scientific). DNA restriction fragments were separated in a PFGE apparatus at 14°C, 6 V/cm, for a specific time for each species (19–23 h). The gel was stained with gel-red and visualized with a UV system.