Trans blot turbo device

Western blot experiments were performed by loading 10 μg of protein (determined by Bradford Assay) in 10% acrylamide SDS–PAGE gels. After separation, proteins were transferred to a nitrocellulose membrane using a Trans‐Blot Turbo device (Bio‐Rad, USA). Non‐specific binding sites were blocked using a 5% BSA solution (diluted in TBS pH 7.5 + 0.2% Tween 20) for two hours, and then, membranes were incubated with the primary antibody overnight at 4°C. Afterwards, blots were washed three times with TBS + 0.2% TWEEN, incubated with HRP‐conjugated secondary Ab (DAKO) for two hours, and again washed. Signal was acquired using ECL detection kit (Thermo Fisher Scientific, 32106) with a MicroChemi 4.2 device (DNR Bio Imaging System). For quantification, Gel‐analyser Software 2010a was used.

The cells were lysed either in RIPA‐buffer [150 mM sodium chloride, 50 mM Tris, 2 mM EDTA, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate SDS, 0.5 mM DTT, Complete ultratablet protease inhibitor, (Roche)] or in SDS‐buffer (150 mM Tris, pH 6.8, 1.2% SDS, 30% glycerol, 15% β‐mercaptoethanol). Protein concentrations of the extracts were determined spectrophotometrically (Pierce BCA Protein Assay Kit, Thermo Scientific). 10–20 μg of protein was loaded to a 12% SDS–PAGE gel followed by a transfer to PVDF membrane (Immobilon, Millipore) either by electroblotting in transfer buffer (20% MetOH, 250 mM Tris, 1.9 M glycine) 100 V for 1 h at 4°C or by Transblot Turbo device (Bio‐Rad) using manufacturer's instructions. After blocking (5% BSA in 0.1% TBS‐Tween), the membrane was probed with the primary antibodies o/n at 4°C. After several washes, the membrane was probed with horseradish peroxidase‐conjugated secondary antibodies, washed again and finally visualized using the SuperSignal West Pico kit (Thermo Scientific) or with Clarity Western ECL (Bio‐Rad).

1 × 10⁶ EVs for each condition shown in Supplementary Fig. 1B were processed for Western blot analysis as follows. iEVs were counted by NTA and lysed at 70 °C for 5 minutes in 5 × loading dye buffer in a total volume of 20 µL. 15 µL samples were loaded into a 4–20% mini protean TGX gel (Bio-Rad). Precision Plus Protein Standard was used as a marker for weight (Bio-Rad). Gel was ran at 100 volts for 75 minutes and then transferred to 0.2 µm PVDF membrane using a Trans Blot Turbo device (Bio-Rad). Wester blotting protocol from Cell Signal Inc. was followed for antibody detection. Primary antibodies rat anti-mouse CD63 monoclonal antibody (Cat#564222, BD Biosciences) and hamster anti-mouse CD81 monoclonal antibody (Cat#559519) were both used at 1:1,000 dilution. Horseradish peroxidase-labeled rabbit antibodies to rat/hamster IgG were used at 1:2,500 dilution. Bound antibodies were revealed with Clarity Western ECL substrate (Bio-Rad). Blot was exposed for 10 minutes with Blue Devil autoradiography film (Genesee Scientific).

Cells were lysed by heating at 100 °C in Laemmli sample buffer (BioRad) and resolved by 10% or 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes using a Transblot Turbo device (BioRad) and blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% skimmed milk powder (blocking buffer). An HRP anti-human FcεRIα was incubated overnight at 4 °C in blocking buffer on a rocking platform. Proteins were detected using Cytiva Amersham™ ECL™ Prime Western Blot detection reagent and ChemiDoc MP imager (BioRad). The signal bands were quantified using BioRad’s Image Lab software against the total protein content.

Cells were lysed using a radio immunoprecipitation assay (RIPA) buffer (Boster, Wuhan, China) containing a protease inhibitor. The total protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA) following the procedure outlined in the instructions. Proteins were separated using 12.5% polyacrylamide gel and transferred to 0.45 μm of polyvinylidene fluoride (PVDF) membranes using a Trans-blot Turbo device (Bio-Rad, Hercules, CA, USA) at 75 V on ice for 1.5 h. The membranes were blocked with 5% skimmed milk (Solarbio, Beijing, China) for 1 h at room temperature. Then, the primary antibody was used to incubate the membranes overnight at 4 °C. The primary antibody and dilution rate are described as follows: anti-NRAMP1 (1:500) (Novus, Wuhan, China), β-actin (1:20,000) (Proteintech, 66009-1-Ig, Rosemont, IL), caspase-3 (1:1000) (Abclonal, Wuhan, China), proliferating cell nuclear antigene (PCNA) (1:5000) (Elabscience Biotechnology Co. Ltd., Wuhan, China). Next, the membranes were incubated with the secondary antibody horse radish peroxidase (HRP) goat anti-rabbit IgG (1:10,000) and HRP goat anti-mouse IgG (1:10,000) (ABclonal, Wuhan, China) at room temperature for 1 h. Finally, the membranes were scanned using chemiluminescence imaging software (AI600 Control. RDP).

Twenty‐five milligrams of muscle biopsies were homogenized with RIPA lysis buffer (Millipore, Temecula, CA) with added Halt™ protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, MA). After centrifugation, supernatants were collected and total protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein were boiled in Laemmli buffer at 95°C for 5 min. 20 μg of protein was separated by SDS‐PAGE and transferred to PVDF membranes (Bio‐Rad Laboratories, Inc., CA) using the semidry Trans‐Blot Turbo™ device (Bio‐Rad). Membranes were incubated with the following primary antibodies, total p62, Sestrin1 and 3 (Abcam, ab56416, ab103121, and ab97792, respectively), Sestrin2 (ProteinTech, 10795‐1‐AP), and p62^Ser403 (GeneTex, GTX128171) (all at 1:1000 dilution, except Sestrin1 which is at 1:100) overnight, and the appropriate anti‐rabbit or anti‐mouse secondary antibodies (Jackson ImmunoResearch Laboratories, PA) linked to horseradish peroxidase (1:10,000) for 1 h at room temperature. The membranes were exposed on a ChemiDoc image device (Bio‐Rad) using enhanced chemiluminescence reagent (ECL Select kit; GE Healthcare Ltd., Little Chalfont, UK). Bands were quantified using ImageJ software (NIH, Bethesda, MD). Western blot data were normalized to the housekeeping protein GAPDH (Abcam, ab36840) (1: 10,000).

U266B1 cells (2 × 10⁶) were lysed in NP40 lysis buffer supplemented with metallo-protease and protease inhibitor cocktails (Sigma-Aldrich, St. Louis, MO, USA). After a Bradford quantification, 40 µg of the whole protein pool was loaded into a 4–20% mini protean TGX gel (Bio-Rad, Hercules, CA 94547, USA). The gel was run at 100 volts for 75 min and then transferred to 0.2 µm PVDF membrane using a Trans-Blot Turbo device (BioRad, Hercules, CA 94547, USA). The Western blotting protocol was followed for antibody detection using specific monoclonal antibodies against different proteins. The following primary antibodies all from OriGene (Austin, TX, USA) were used: mouse monoclonal antibody to human SOX4 (Cat # TA324704), AKT1 (Cat # TA504230), PI3KCA (Cat # AM06736PU-N), HIF1-A (Cat # AM06608SU-N), and rabbit monoclonal antibody to PTEN (Cat # AP15248PU-N), pAKT1 (Cat # TA313266), TGFβ1 (Cat # TA506585). Bound antibodies were revealed with Clarity Western ECL substrate (Cat # 1705061, BioRad, Hercules, CA 94547, USA). A ChemiDoc™ Imaging Systems (BioRad, Hercules, CA 94547, USA) was used as an imaging system and band quantification was performed using the ImageLab software.