Enhanced chemi luminescence (ecl)

Cells were collected and lysed with RIPA buffer (50 mM Tris, pH 7.2, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl₂ and 5% glycerol) containing 1% protease inhibitor PMSF. Protein concentration was determined using BCA Protein Assay Kit (#P0011, Beyotime). Proteins (25 μg) from each lysate were separated by electrophoresis in a 10% polyacrylamide resolving gel and transferred onto a polyvinylidene difluoride (PVDF) membranes. After blocking with 3% BSA (#ST023, Beyotime) in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) at room temperature for 1 h, membranes were incubated at 4 °C overnight in TBS-T containing following antibodies against TWIST1 (#sc-81417, Santa Cruz; CA, USA), Tubulin (#2146 s, Cell Signaling Technology; Beverly, MA, USA), O-GlcNAc (#ab308178, Abcam) and OGT (#ab177941, Abcam) followed by the addition of secondary antibody conjugated with HRP (#A0208, #A0216, Beyotime). Bands were visualized by enhanced chemiluminescence (ECL; Vazyme Biotech; Nanjing, China).

MDCK cells were infected with the H1-H16 subtype corresponding recombinant viruses or 293 T cells were transfected with pCAGGS-H17HA or pCAGGS-H18HA plasmid. Cells were collected and lysed with NP-40 lysis buffer (Beyotime, Shanghai) after 24 h’ infection or transfection. Cell lysates were mixed with 4 × loading buffer (Solarbio, Beijing) and denatured at 100 °C for 15 min, then were separated using SDS-PAGE with a 10% polyacrylamide gel [44 ] and transferred to NC membranes (GE Healthcare, Amersham). Next, membranes were blocked with 5% skimmed milk in PBS at 37 °C f for 1 h, then washed six times (5 min per time) with PBST and incubated with primary antibody 3C12 (1:800) at 4 °C overnight. Then, after six times washing, the membrane was incubated with HRP-conjugated goat anti-mouse antibody (1:10000; KPL, Gaithersburg, MD) as secondary antibody at 37 °C for 1 h. Membranes were then washed and the target protein bands were detected with enhanced chemiluminescence (ECL) (Vazyme, Nanjing) and the signals were recorded using Image Lab Software (Bio-Rad). GADPH served as a loading control.

After each treatment, the cell pellets were lysed in ice-cold RIPA buffer (Solarbio) containing PMSF. The cell lysates were centrifuged at 12,000 rpm at 4 °C to produce the whole cell extracts, and the protein concentration was quantified using the BCA protein assay kit (Solarbio). Sample loading buffer was added and boiled at 95 °C. Equal amounts of protein were separated on a 10% SDS-PAGE and subsequently transferred to PVDF membranes (Millipore). After blocking with 5% non-fat milk, the membranes were incubated at 4 °C overnight with primary antibodies against ZO-1 (1:300), claudin-5 (1:300) or GAPDH (1:5000), and further incubated with horseradish peroxidase-conjugated secondary antibodies. The protein bands were washed and developed with enhanced chemiluminescence reagents (ECL, Vazyme Biotech, Nanjing, China) and scanned with the Kodak Digital Imaging System. The optical density (OD) values of each band were normalized to GAPDH using Image J software (version 1.37, NIH, Bethesda, MD, USA).

Total proteins were extracted from cells with precooled RIPA lysis buffer (C1053, APPLYGEN, Beijing, China), and the concentration of the whole proteins was quantified with Enhanced BCA Protein Assay Kit (P0010, Beyotime Institute of biotechnology, Shanghai, China). After boiling with 4 × loading buffer for 5 min, equal amounts of denatured protein in each sample (20 μg) were prepared for electrophoresis on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The immunoblot was blocked with 5% skim milk for 1.5 h at room temperature, followed by overnight incubation with primary antibodies (both diluted 1:1,000) at 4°C. After washing with Tris-buffered saline 0.1 Tween 20 (TBST), the membranes were incubated with species-specific HRP conjugated secondary antibodies (diluted 1:10,000) at room temperature for 2 h. The HRP signals were detected with enhanced chemiluminescence (ECL, E412-02, Vazyme, Nanjing, China) substrate after washing with TBST and quantified by ImageJ after developing. Moreover, β-actin was applied as the reference protein. Each experiment was performed in triplicate.

The size distribution and zeta potential of the EVs were analyzed by dynamic light scattering (DLS) using Nano-ZS ZEN3600 (Malvern, UK).
For morphology observation, 30 μL of the purified EVs were dropped on the carbon film covered copper grids, dried for 5 min, stained with 2% uranyl acetate for 1 min, and observed under transmission electron microscopy (TEM, H-7650, Hitachi, Tokyo, Japan) at 80 kV.
For biomarker analysis, EV positive and negative markers were examined by Western blotting using the following antibodies: CD63 (1:1000, ab134045), TSG101 (1:1000, ab83, Abcam, Cambridge, MA, USA)), CD81 (1:1000, sc-23962, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Alix (1:1000, 2171S), and Calnexin (1:1000, 2679S, Cell Signaling Technology, Beverly, MA, USA). A total of 30 μg EV samples were boiled at 95 °C for 10 min, separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked with 5% (w/v) non-fat milk in TBST at room temperature (RT) for 1 h, incubated overnight with the primary antibodies above, followed by incubation with HRP-conjugated secondary antibodies (1:5000, Beyotime). Bands were visualized by enhanced chemiluminescence (ECL, Vazyme Biotech, Nanjing, China).

Left ventricular samples peri against infarction and cell samples were prepared in lysis buffer containing 1 mM PMSF. Equal amounts of protein samples were separated by denaturing SDS–PAGE, followed by transferring to polyvinyl difluoride (PVDF) membranes (Millipore Corporation, Bedford, MA, United States). The membranes were blocked with 3% BSA and stained with appropriate primary antibodies against GAPDH, β-Actin, Drp1, p-Drp1, Mfn2, CACNA1C, CaMKII, p-CaMKII, NOX2, p67^phox and NOX4 (dilution 1:8000, 1:1000, 1:1000, 1:500, 1:1000, 1:800, 1:2000, 1:2000, 1:5000, 1:5000, 1:2000, respectively) for 16 h at 4°C. After washing, membranes were probed with the HRP-conjugated secondary antibodies (dilution 1:8000, Bioworld, Louis Park, MN, United States). The antigen-antibody complexes were detected using enhanced chemiluminescence (ECL, Vazyme Biotech, Nanjing, China) and visualized by ChemiDoc^TM MP System (Bio-Rad). The results were quantified using Image Lab^TM software (version 4.1, Bio-Rad, Hercules, CA, United States).

Fresh kidney samples from each group were ground with grinding rods and lysed with RIPA buffer (Beyotime, China) containing PMSF and protease inhibitors (APExBIO). After incubation on ice for 30 min, the supernatant was obtained by centrifugation at 12,000 g for 10 min at 4°C. The protein concentration was determined by the BCA method. Then, the samples were loaded onto a gel for electrophoresis. The proteins in the gel were then transferred to a PVDF membrane, which was blocked with 5% skim milk in TBST at 37°C for 1 h, followed by incubation at room temperature with Col-I, α-SMA, TGF-β, Rab7, LC3B, BECLin-1, or GAPDH primary antibodies for 90 min. The membrane was washed twice with TBST for 15 min, and then incubated with goat anti-rabbit secondary antibodies at room temperature for 1 h. After the membrane was washed twice with TBST for 15 min, the blots were developed with ECL (Vazyme), and the results were analyzed with ImageJ software (Media Cybernetics Inc., USA).