Ripa buffer

Whole cell lysates were prepared using a RIPA buffer (Sigma-Aldrich), sonicated, and cleared of cellular debris by centrifugation at 14,000g for 15 min. TA muscles were snap-frozen in liquid nitrogen and homogenised with RIPA buffer (Sigma-Aldrich) using a DUALL Tissue Homogeniser (Kimble Kontes). Tissue was centrifuged at 3,000g for 10 min, and then 14,000g for 15 min prior to measuring concentrations of protein in the lysates using the Bradford Protein Assay (Thermo Fisher Scientific, Waltham, MA, United States). Western blot analyses were performed as described previously (Gao et al., 2008 (link)) using nitrocellulose membranes (Biorad; Hercules, CA, United States) and commercial antibodies to Myosin (Merck, Darmstadt, Germany). Equal protein loading and electroblot transfer were verified by staining the membrane post transfer with Ponceau S Red (Sigma-Aldrich). This method has been routinely used (Perry et al., 2018 (link)) and validated as a reliable measure of protein loading with immunoblotting (Romero-Calvo et al., 2010 (link)). Following application of Chemiluminescent Substrate (SuperSignal^TM West Pico PLUS, Thermo Fisher), densities of detected protein bands were recorded with a BioRad chemiluminescence imager (ChemiDoc XRS+, BioRad) and densities determined using Image J (NIH, Bethesda, MD, United States).

Cells were lysed in 100 μL RIPA buffer (Sigma-Aldrich) with sodium orthovanadate (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich cat # P8340) added just prior to lysis, and the concentration of protein in the lysates was quantified using a Bio-Rad protein assay (Bio-Rad) using a spectrophotometer (abs: 595). Equal amounts of protein lysate were subject to electrophoresis and Western analysis. For co-immune precipitation, cells were lysed in 100–200 μL modified RIPA buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1% Tergitol NP-40 (Sigma-Aldrich)) with protease inhibitors added prior to lysis as above. Precleared lysates were immunoprecipitated overnight with GFP-Trap agarose beads (Allele Biotech, San Diego, CA, USA) or primary antibody as appropriate and washed 3 × 3 min with lysis buffer, then Western blotted as above.

Whole protein extraction from 3D cultured NP cells was performed using radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich). NP cell pellets were washed twice in cold PBS (2500 x g, 5 min) and resuspended with 300 ml cold RIPA buffer containing protease inhibitor and phosphatase inhibitor cocktails (Sigma-Aldrich). After sonication (30 sec, 50% pulse) the mixture was shacked gently on ice (15 min) and centrifuged (14000 x g, 15 min and 4 °C). Supernatants were transferred to new tubes for protein quantification. Total protein concentration in samples was determined by using Pierce Micro BCA Protein Assay Kit according to the instruction manual (Thermo Scientific).

Brain homogenates were spun at 1000×g for 10 min at 4 °C [20 (link)]. The supernatant (cortical fraction) was spun at 14,000×g for 10 min. The resulting cell pellet was resuspended and digested in RIPA buffer (Sigma) containing protease inhibitor cocktail (PIC, Sigma) for 1 h on ice. The MV pellet was resuspended and digested in a mixture of RIPA buffer (Sigma) and protease inhibitor cocktail (PIC, Sigma) for 1 h on ice. All homogenates were sonicated on ice for 90 s and spun at 14,000×g for 10 min at 4 °C for final collect of the protein lysate. Total protein was quantified by bicinchoninic assay (BCA, Pierce) and 25 µg was loaded onto a 4–20% Criterion TGX Precast Protein Gel (Bio-Rad). Protein was transferred onto a nitrocellulose membrane and probed for the neuronal marker TUJ1 (Abcam, 1:1000, ab18207) or the brain MV marker von Willebrand factor (Abcam, ab174290, 1:1000) and cyclophilin B as a loading control (Abcam, ab16045, 1:1000). Corresponding secondary antibody conjugated to HRP was used (1:5000, GE Amersham) and bands were visualized using ECL reagents (Perkin-Elmer) on an Amersham Imager 600 (GE Amersham).