Nupage 4 12 bis tris precast gel

Washed cells or sEV‐enriched pellets were lysed in lysis buffer (1% Triton‐X100, 1 mM EDTA, 150 mM NaCl, 20 mM Tris pH 7.5) supplemented with 1× cOmplete™ EDTA‐free Protease Inhibitor Cocktail (11836170001, Roche) on ice for 30 min with regular vortexing. Lysates were centrifuged at 14,000×g for 15 min at 4°C. Resultant lysates were mixed with 4× NuPage LDS sample buffer (ThermoFisher), boiled at 65°C or 95°C for 10 min and subjected to SDS‐PAGE on NuPage 4%–12% Bis‐Tris precast gels (ThermoFisher). Samples were transferred to PVDF membranes, blocked with 5% milk/PBS‐Tween and probed with the above primary and secondary antibodies. To visualise proteins, membranes were imaged using an Odyssey CLx (Li‐Cor).

Cells were lysed in standard lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EGTA, 50 mM NaF, 5 mM Na₄P₂O₇, 1 mM Na₃VO₄, and 10 mM ß-glycerol phosphate in the presence of protease inhibitors, cOmplete Mini, Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Then, proteins were separated using NuPAGE 4–12% Bis-Tris pre-cast gels (Thermo Fisher Scientific) and transferred into nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% milk in TBST, incubated overnight at 4°C with primary antibodies diluted 1:1000, and then incubated for 1 hr with HRP-linked secondary antibodies at room temperature. Finally, proteins were detected by chemiluminescence. The following primary antibodies were used: BRD4 (ab128874, Abcam), Cas9 (7A9-3A3, 14697, Cell Signaling Technologies), CHD4 (A301-082A, Bethyl Laboratories), Flag (clone M2, F1804, Sigma Aldrich), FOXO1/FKHR (H-128, sc-11350, Santa Cruz Biotechnology), GAPDH (14C10, 2118L, Cell Signaling Technologies), HDAC1 (10E2, 5356, Cell Signaling Technologies), HDAC2 (3F3, 5113S, Cell Signaling Technologies), MTA2 (M7569, Sigma Aldrich), and RBBP4 (A301-206A-M, Bethyl Laboratories).

Protein samples were obtained by tissue lysis with ice-cold RIPA buffer (Sigma, St Louis, MO USA) and Proteinase Inhibitor Cocktail (Roche). A total of 20 μg of protein was separated on NuPAGE 4–12% Bis-Tris precast gels (ThermoFisher, Waltham, MA USA) and transferred to nitrocellulose membranes. The membranes were blocked for 1 h in 5% non-fat milk and successively blotted overnight at 4°C in 5% milk with a primary SMN-specific antibody (ab610646,BD Biosciences, USA, 1:2000) and a primary GAPDH antibody (ab8245, Abcam, USA, 1:5000).
Membranes were washed and incubated with specific secondary antibodies for 1 h at room temperature (anti-Mouse 1:5000). Protein bands were detected with ECL Western blotting substrate (ThermoFisher, Waltham, MA, USA) followed by exposure to X-ray film (Kodak) or to UVItec CAMBRIDGE Alliance.

Cells were lysed with cell lysis buffer (Cell-Signaling Technology), and the extracted protein fraction was quantified by Bradford protein assay (Bio-Rad laboratories, Hercules, CA, USA) to ensure equal loading of 50 µg of protein for each sample. Samples were loaded into NuPAGE® 4%-12% Bis-Tris Precast Gels (Thermo Scientific, UK) for electrophoresis. Protein bands were transferred from gel onto polyvinylidenedifluoride (PVDF) membranes, and incubated with primary antibodies-Grp78, GADD153 or cleaved caspase-3 p17 (H-60) (rabbit polyclonal antibody; Santa Cruz Biotechnology, UK) at dilution factor of 1:300. On the second day, membranes were incubated with secondary antibody (anti-rabbit HRP conjugated, Cell-Signaling Technology) for 1 h at room temperature at dilution factor of 1:1000, before development with enhanced chemiluminescence (ECL) system (Santa Cruz, Dallas, TX, USA). Protein bands were visualized by Syngene GeneSnap software (Syngene, Cambridge, UK). The intensity of grey scale of protein bands was assessed with ImageJ.