Na931 1ml

Total protein was isolated from mid-exponentially growing yeast by rapid capture of protein expression through 5% tricarboxylic acid treatment for 10 min, followed by a wash in acetonitrile. The cell pellets were then dried at room temperature for 30 min before bead-beating in Tris-acetate-EDTA buffer for 5 min at room temperature. Samples were then resuspended in SDS loading buffer from NuPage, boiled for 5 min, and loaded on 4–12% polyacrylamide Bis-Tris gels and separated by electrophoresis in MOPS buffer. Proteins were then transferred to a nitrocellulose membrane and were blocked for 1 h in TBST (Tris-buffered saline, 0.1% Tween 20) with 5% bovine serum albumin. Primary antibodies (DYKDDDDK Tag Antibody, Cell Signaling Technology 2368S; α-Pab1 Antibodies-Online ABIN1580454 (clone 1G1)) were incubated with membranes at a 1:1,000 dilution in TBST for 1 h at room temperature, washed with TBST, then incubated for 30 min at room temperature with anti-rabbit (Cell Signaling Technology, 7074S) and anti-mouse (Cytiva NA931-1ML) HRP-linked antibodies at a 1:10,000 dilution. Membranes were developed with Pierce ECL western blotting substrate and imaged on the chemiluminescence channel on a ProteinSimple instrument.

The samples of FF-sEVs prepared with adjusted amounts of protein were loaded onto polyacrylamide gels for electrophoretic separation of proteins at 20 mA. After blocking with Skim Milk (Snow Brand Megmilk Co., Japan) or Blocking One (Nacalai Tesque Inc., Japan) for 1 h at room temperature, the membranes were incubated overnight at 4 °C with the following primary antibodies: mouse monoclonal anti-CD9 (CBL162; Merck; dilution 1:100), rabbit monoclonal anti-CD63 (EXOAB-CD63A-1; System Biosciences, LLC, CA, USA; dilution 1:1000), mouse monoclonal anti-CD81 (sc-166029; Santa Cruz Biotechnology, TX, USA; dilution 1:100), and mouse monoclonal anti-GRP (sc-393402; Santa Cruz Biotechnology; dilution 1:100). The membranes were subsequently washed three times for 5 min each using Tris-buffered saline with 0.1% Tween® 20 (TBST) and incubated for 1–3 h at room temperature with secondary HRP-conjugated mouse anti-rabbit IgG (NA934-1ML; Cytiva Lifesciences, USA; dilution 1:5000) or anti-mouse IgG (NA931-1ML; Cytiva; dilution 1:2000) antibodies. The protein ladder is MagicMark^TM XP Western Protein Standard (Thermo Fisher Scientific). The membranes were imaged using ImageQuant LAS 4010 (GE Healthcare, IL, USA). The uncropped blots are shown in Supplementary Fig. S2.

Proteins were extracted using an AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. Protein concentrations were quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific) at 562 nm using a PerkinElmer EnSpire plate reader. Proteins (20 µg) were mixed with NuPAGE 4× lithium dodecyl sulfate sample buffer (Invitrogen) and NuPAGE 10× reducing agent and then heated at 70 °C for 10 min before centrifugation at 17,500 rpm for 10 min at 4 °C. Proteins were separated on a 4–12% Bis-Tris-polyacrylamide gel (Invitrogen) and transferred to an iBlot Mini Transfer Stack PVDF membrane (Invitrogen). The membranes were probed with antibodies against UCHL1 (1:2000; MAB6007; R&D Systems, Minneapolis, MN, USA) or β-actin (1:5000; MAB1501R; Millipore, Bedford, MA, USA) overnight at 4 °C after blocking with 5% skim milk in Tris-buffered saline Tween 20 for 1 h. The secondary antibody anti-mouse IgG, HRP-linked (1:1000 or 1:2000; NA931-1ML, Cytiva, Westborough, MA, USA) was used. Immune complexes were visualized using chemiluminescence (EMD Millipore, cat. no. WBLUF0100, cat. no. WBLUC0100) and quantified using a ChemiDoc XRS Plus system with Image Lab software (Bio-Rad, Hercules, CA, USA). The primary antibodies used in this study are listed in Table 2.