Xcell surelock mini cell system

Soluble fractions were thawed on ice and mixed with 4× NativePAGE sample buffer comprising 50 mM Bis-Tris, 50 mM NaCl, 10% (w/v) glycerol, and 0.001% Ponceau S, pH 7.2. Ten-microgram samples per well were loaded onto NativePAGE Novex 3–12% BisTris gels (ThermoFisher). The proteins were fractionated for 90–115 min at 150 V using an XCell SureLock Mini-Cell system (ThermoFisher, Carlsbad, CA). The fractionated proteins then were transferred to PVDF membranes for 1 h at 25 V using the XCell II™ Blot Module (ThermoFisher). After the transfer, the membranes were incubated in 20 mL of 8% acetic acid for 15 min to fix the proteins. The membranes were probed with anti-human tau antibody HT7 (ThermoFisher) or anti-phospho-tau antibody AT8 (ThermoFisher). The intensity of the protein bands was quantified densitometrically using Image J [31 ].

Purified rabbit anti‐SosA antibody was obtained via GenScript Biotech (Netherlands). A custom peptide, CEHKDPTNNSEQRDGA, comprising AAs 57–70 of SosA was used for immunization. Bacterial cells were pelleted and frozen immediately at −80°C before being resuspended in PBS containing cOmplete™ Mini Protease Inhibitor Cocktail (Sigma‐Aldrich) and lysed by bead beating (Fastprep‐24™, MP Biomedicals). The protein concentration of the lysates was measured using the Qubit™ Protein Assay Kit (ThermoFisher Scientific) and samples were normalized to equal amounts of protein, 20 µg of total protein was separated on NuPAGE^® 4‐12% Bis‐Tris gels using MES buffer and the XCell sure‐lock mini‐cell system (ThermoFisher Scientific), transferred to a polyvinylidene difluoride membrane (PVDF) and probed with primary antibody diluted 1:1000. Bound antibody was detected with the WesternBreeze^® Chemiluminescent Kit, anti‐rabbit according to the instructions from the manufacturer (ThermoFisher Scientific). All western blots were independently repeated with similar results. Uncropped versions of blots are displayed in Fig S5.

Male and female aVICs and qVICs were cultured as described above for 48 h, at which point cell lysates were collected using Pierce RIPA buffer. A microBCA assay (ThermoFisher) was conducted to determine protein concentration for each sample. Samples were diluted to the same protein concentration and then denatured in a 70°C water bath for 10 min with Novex™ Tris-Glycine SDS Sample Buffer (LC2676; ThermoFisher). The samples were loaded into a 4–12% Tris-glycine gel and electrophoresis performed using a XCell SureLock Mini-Cell system (EI0001; Thermo Fisher Scientific) was run at 200 V for 30 min. The gel was blotted onto a PVDF membrane overnight at 4°C at 16 V. The membrane was blocked in 5% w/v milk in PBST for 90 min at room temperature and then incubated with anti-chondromodulin-1 antibody (MAB41681, R&D) at a dilution of 1:1,000 at 4°C overnight. The membrane was then washed in TBST and incubated with an HRP conjugated secondary antibody at a dilution of 1:10,000 for 1h at room temperature, followed by washing with TBST, exposing to the ECL substrate, and imaging on the Bio-Rad ChemiDoc XRS imaging system (1708265; Bio-Rad).

Extracted proteins were separated by SDS‐Page using the Xcell sure lock mini‐cell system (Thermo Fisher Scientific), and then transferred onto PVDF membranes (Immobilion‐P, Merck Millipore, Watford, UK). Membranes were then incubated for 1 hr at room temperature in blocking medium (5% non‐fat milk dissolved in TBST [0.05 M Tris, 0.15 M NaCl, pH 7.2, 0.1% (v/v) Tween20]), before incubation in primary antibodies overnight at 4°C with constant agitation. After washing in TBST membranes were incubated in secondary antibodies for 1 hr at room temperature. All antibodies were diluted in blocking medium (see Supporting Information Table S1 for details of antibodies).

The ambient pressure reactions were carried out in a thermoshaker and the HHP reactions in a home-built high-pressure vessel. The reaction products were analysed by denaturing urea polyacrylamide gel electrophoresis (urea PAGE) using a XCell SureLock Mini-Cell system (ThermoFisher) with Novex 15% TBE-Urea gels (ThermoFisher) and TBE running buffer (Ambion). After electrophoresis, the gels were directly stained with SYBR Gold (Molecular Probes) and the bands were analysed by an ultraviolet transilluminator (AlphaImager Mini, ProteinSimple). The intensity of each RNA fragment band was quantified using ImageJ software (N.I.H., USA) and the reaction product is presented by the percentage of cleaved RNA over the total RNA amount (for details, see the Supplementary Information). Hence, the PAGE results take into account the native and cleaved states of the HpRz, only, and, in stark contrast to the FRET data, do not contain any information on undocking conformations and/or dissociation steps.