Bis tris gel

Protein extract was obtained by lysing PBMC or purified CD3 cells using Mammalian Protein Extraction Reagent (Thermo Scientific) supplemented with 0.1% SDS and Halt Protease and Phosphatase Inhibitor. Twenty μg (20μg) of protein was loaded into an 8% Bis-tris gel (Life Technologies) for detection of Arg1, or a 4-12% Bis-tris gel for detection of CD3ζ. After transfer to a PVDF membrane it was blocked with 5% milk for 1h and incubated overnight with appropriate primary antibodies. Primary anti-human antibodies included arginase-1 (BD Biosciences), phospho CD3ζ (Abcam), CD3ζ (Abcam), and β-actin. Following incubation HRP was detected using ECL Western Blotting Substrate (Thermo Scientific).

To establish the purity of the BDEVs, we examined the expression of positive markers: Hsp70 (SAB4200714, Sigma-Aldrich, St. Louis, MO, USA), Flot-1 (abcam41927, Abcam, Cambridge, UK), CD81 (MCA1846, Bio-Rad Laboratories, Inc., Hercules, CA, USA), CD63 (BD551458, BD Biosciences, Franklin Lakes, NJ, USA), HSP90 (C45G5, Cell Signaling Technology, DANVERS, MA, USA), and negative marker GM130 (BD610822, BD Biosciences, Franklin Lakes, NJ, USA), as described in previous papers [11 (link),14 (link),15 (link)]. Briefly, 25–30 μg of BDEV was subjected to Western blot under reducing (Hsp70, Flot-1, GM130) and non-reducing (CD81, CD63, HSP90) conditions using 4–12% Bis-tris gels (Invitrogen, Waltham, MA, USA).
For YWHAH validation, 15 μg of BDEV was subjected to Western blot under reducing conditions using 10% Bis-tris gel (Invitrogen, Waltham, MA, USA) against YWHAH (15222-1-AP, Proteintech, Rosemont, IL, USA). Primary and secondary antibody dilutions were carried out according to the manufacturer’s suggestions. Blots were developed using Azure CSeries Imager (Azure Biosystems, Dublin, CA, USA) with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA).

Denatured proteins were resolved using either 4–12% Bis-Tris gels or 10–20% tricine gels (ThermoFisher) according to manufacturer’s recommendations. Proteins resolved on Bis-Tris gels and tricine gels were transferred respectively onto 0.45 μm and 0.22 μm nitrocellulose membranes (ThermoFisher) and probed with appropriate primary antibodies (Additional file 1: Table S3). Secondary antibodies conjugated with IRDye® 800CW or 680RD (LI-COR Biosciences) were used and images captured with the Odyssey® infrared imaging system (LI-COR Biosciences). For densitometric quantification, the intensity of the bands was determined using Image Studio™ Lite software (LI-COR Biosciences).

Mitochondria-enriched and cytosolic fractions were isolated from cortex, hippocampus, cerebellum and olfactory bulb of WT and Wdfy3^+/lacZ mice as described before^{68 (link)}. Thirty-five µg of proteins were solubilized in SDS sample buffer (Life Technologies, Grand Island, NY) and loaded onto a 4–12% bis-tris gel (Life Technologies) as previously described^{68 (link)}. After transferring proteins with an iBlot apparatus (Life Technologies), membranes were blocked with LI-COR blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at 20 °C and subsequently probed overnight at 4 °C with the following antibodies: anti-Lamp2 (Abcam, Cambridge, MA; 1:1,000 dilution), anti-LC3 (Novus Biologicals, Littleton, CO; 1:1,000 dilution), anti-Mfn2 (Proteintech, Rosemont, IL; 1:500 dilution), anti-MnSOD (Millipore, Billerica, MA; 1:1,000 dilution), anti Sqstm1 (Cell Signaling Technology, Danvers, MA; 1:500 dilution), anti-Park2 (Abcam; 1:500 dilution), and anti-Pink1 (Novus Biologicals; 1:1,000 dilution). As a loading control, we used anti-β-actin antibody (Sigma, St. Louis, MO; 1:20,000 dilution, 1 h at 20 °C). Secondary antibodies were from LI-COR (Lincoln, NE; 1:10,000 dilution). Membranes were visualized with the use of the Odyssey Infrared Imaging System (LI-COR) and densitometry analysis carried out with ether the Carestream or ImageJ softwares.

Cell pellets were lysed in whole cell lysis buffer (20 mM HEPES KOH (pH 7.4), 50 mM NaCl, 2%w/v NP40, 0.5%w/v NaDeoxycholate, 0.2%w/v SDS, 1 mM NaOrthovanadate, 1 mM EGTA pH 7, 10 mM NaF, 1 mM PMSF, protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined by BCA Assay (Thermo-Fisher Scientific, Loughborough, UK). Samples were added to 4× Gel loading buffer (Life Technologies), DTT (0.083M), heated to 70°C for 5 min. Secreted proteins were concentrated from supernatents using Amicon Ultra 10K filters (Millipore) and equal volume of concentrated supernatents were added to 2XSDS loading buffer, heated to 95°C for 5 min. Samples were resolved on a 4–12% Bis-Tris gel (Life technologies, Paisley, UK) and transferred to a nitrocellulose membrane and blocked in 5% PVP, 0.5% FBS. Membranes were incubated with antibodies (Text S2) and detected by ECL plus (GE, Buckinghamshire, UK).