Ab7291

RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) with protease inhibitor (P0100, Solarbio life sciences, Beijing, China) was added to rat myocardial tissue or H9C2 cells to extract protein according to the manufacture’s protocol. The protein concentration was detected using a BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). Protein samples, which were mixed with loading buffer and heated at 100°C for 8 min, were separated by 12% SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF) pre-activated with methanol. The membrane was then blocked with 5% bovine serum albumin (BSA) for 1 h and immersed in the primary antibody solution overnight at 4°C. The primary antibodies were anti-NRF2, anti-HO-1 (E3F4S, CST, United States), anti-PKC (ab23511, Abcam, United Kingdom), anti-PKC (phosphor T497, ab59411, Abcam, United Kingdom), anti-α-tubulin (ab7291, Abcam, United Kingdom), and anti-H3 (17168-1-AP, Proteintech, Wuhan, China). The membranes were incubated with secondary antibodies (ZB-2301 or ZB-2305, ZSGB-BIO, Beijing, China) and the protein signals were detected using enhanced chemiluminescence (ECL) detection system. Images were analyzed using Image J software (National Institutes of Health, United States).

Exosomes in ERCMs obtained from AMMSCs were labeled with red CM-DiI membrane dye (C7000; Invitrogen) and prepared at 4 × 10¹⁰ particles/mL [45 (link),49 (link),50 (link)].
pRGCs were seeded on 8-well chamber slides (NUNC C7182; Thermo Fisher Scientific, Waltham, MA, USA) at 1 × 10⁵/mL. After 24 h, cells were damaged with 150 µM H₂O₂, and then incubated with labeled exosomes (50 μL/mL) for 4 h at 37 °C in a CO₂ (5%) incubator. The cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Fixed cells were treated with 0.1% Triton X-100 (Thermo Fisher Scientific) at room temperature. Five min later, they were washed in PBS, and blocked with 1% bovine serum albumin (BSA) for 1 h. The cells were immunostained with anti-α-tubulin antibody (1:1000, ab7291; Abcam, Cambridge, UK) for 2 h at 37 °C followed by goat anti-mouse IgG Alexa Fluor^TM 488 (1:500, Invitrogen) for 1 h at room temperature. The cell nuclei were stained with DAPI (Thermo Fisher Scientific) and examined under a microscope (BX51; Olympus, Tokyo, Japan) [45 (link),49 (link)].

Human EGF and bFGF were from Thermo Fisher Scientific (NY, USA). Human TGFβ1 and TNF-α were from R&D systems, Inc. (MN, USA). Human TGFβ2 and TGFβ3 were from Prospec-Tany TechnoGene Ltd. (East Brunswick, NJ, USA). Thrombin, PD98059, SB202190, SP600125 were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Human recombinant VEGF was purchased from Prospect Biotech (Rehovot, Israel). Estrogen and progesterone were purchased from Cayman Chemical Co. (Ann Arbor, Michigan, USA). Recombinant human CTGF was obtained from Thermo Fisher Scientific eBioscience (Waltham, MA, USA). The antibodies (Abs) raised against vimentin (sc-6260), HSP47 (sc-8352) and phospho-Smad2/3 (sc-11769) were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The Ab for total Smad2/3 (#3102) was purchased from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). The Ab for α-SMA (GTX100034) was purchased from GeneTex (Hsinchu City, Taiwan). The Abs for collagen (ab34710), CTGF (ab6992) and α-tubulin (ab7291) were purchased from Abcam (Cambridge, MA). TGFβ isoforms were dissolved in 4 mM HCl/0.1% BSA (vehicle).

To determine the delivered protein in cells and check the leakage of the in-cell NMR samples, each cell sample was centrifuged at 300 g for 3 min immediately after the experiment. Then the supernatants and pellets were resuspended in Laemmli buffer to a final volume of 500 μL, and boiled for 10 min. These samples were diluted by 10 times, and loaded to a 15% SDS-PAGE gel. The primary antibodies used were listed as follows: Tau/k19 (abcam, ab64193, 1:1000), pk19 (abcam, ab75603, 1:1000) and tubulin (abcam, ab7291, 1:1000). Delivered k19 in HEK-293T cells was ~25 μM measured by ImageJ (Available online: https://imagej.net/Welcome) [54 (link)].