Alexa fluor 680

We transfected 293T cells seeded at 2 × 10⁵ cells/well in a 12-well plate with the pHDM-vUTR-WSN-NA-aa1-40-H2B-V5-IRES-GFP constructs or pHDM-vUTR-NP-FLAG constructs. We collected cell lysates 20 hour post transfection, using RIPA buffer containing (1% NP40, 1% Sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM Tris (pH 8), 0.1 mM EDTA, and Roche cOmplete ULTRA Tablet Protease Inhibitor).
For NA43, lysates were run on a 16.5% tris-tricine gel. For NP, lysates were run on a 4-20% tris-glycine gel. NA was detected using 1:2500 dilution of anti-V5 antibody (Invitrogen R960), followed by a 1:2500 dilution of DyLight 800 Rabbit anti-Mouse (Invitrogen SA5-10164). NP was detected using 1:2500 dilution of anti-FLAG antibody (Sigma F1804), followed by a 1:2500 dilution goat anti-mouse Alexa-Fluor 680 (Invitrogen A-21058). For loading controls, we used either GAPDH or H3. We used anti-GAPDH at a 1:2500 dilution (RD systems AF5718), followed by a 1:2500 Alexa-Fluor 680 donkey anti-goat secondary (Invitrogen A-21084). We used anti-H3 at a 1:10000 dilution (abcam 1791), followed by a 1:10000 Alexa-Fluor 680 goat anti-rabbit secondary (Invitrogen A-21109). Western blots were imaged using the LI-COR imaging system.

After sacrifice, LV tissue samples were collected and immediately cryo-preserved in liquid nitrogen and stored at −80 °C. Total protein lysates were prepared as described before⁵⁹ . Equal amounts of proteins were separated on a 4–12% Bis-Tri gradient gel (Invitrogen) and transferred onto polyvinylidene difluoride membrane (Millipore). The membrane was blocked with Odyssey blocking buffer (Li-cor) and probed for lysyl oxidase (LOX), (sc-373995 Santa Cruz), Periostin (ab79946, Abcam), TGF-β1 (ab92486, Abcam), Osteopontin (ab8448, Abcam), Fibroblast activation protein-α (orb97039, Biorbyt) and GAPDH (sc-25778, Santa Cruz). Immunofluorescence detection was performed with matching secondary antibodies (Li-cor 800CW or Alexa Fluor 680, ThermoFisher Scientific) using an Infrared imaging system (Li-cor, CLx).

Transfected cells were incubated in serum-free medium for 2 h to remove any remaining transferrin. Cells were then exposed to 50 μg/ml transferrin conjugated to AlexaFluor 680 (ThermoFisher) at 37 °C for 30 min. Cells were then washed three times with ice-cold PBS and fixed with 4% paraformaldehyde (PFA) for 10 min. After washing the cells at least three times with PBS, transferrin bound on the outside of cells was removed by two 30 min acid washes with 20 mM MES pH 4, 150 mM NaCl, with agitation at 4 °C. Following three washes with PBS, the coverslips were mounted on slides and imaged with a Leica LSM700 confocal microscope.

Cells were lysed in buffer containing 140 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1 % Triton X-100, 0.1 % sodium deoxycholate, and 0.1 % SDS with protease and phosphatase inhibitors (Boston Bio Products). Samples were resolved by SDS–PAGE and transferred onto nitrocellulose membranes. Membranes were blocked in Odyssey Blocking Buffer (LI-COR) for 1 hr and incubated at 4 °C overnight with primary antibodies. Primary antibodies were used as follows: mouse Actin 1:2,500 (ProteinTech Group, 66009-1-Ig), mouse GAPDH 1:1000 (Cell Signaling Technology, 5174 S), rabbit ENAH 1:250 (Sigma, HPA028696), rabbit EVL 1:1000 (Sigma, HPA018849), rabbit VASP 1:1000 (Cell Signaling Technology, 3132 S). Membranes were incubated with secondary antibodies conjugated to either Alexa Fluor 680 or 790 (Thermo Fisher) for 1 hr. Immunoblots were scanned using Odyssey CLx imager (LI-COR).

Proteins separated by electrophoresis were transferred onto PVDF (polyvinylidene difluoride) membrane (Immobilon FL 0.45 µm, Merck) by semi-dry electroblotting using a Transblot SD apparatus (Bio-Rad). After blotting, the PVDF membrane was washed for 5 min in TBS (150 mM Tris-HCl, 10 mM NaCl; pH 7,5) and blocked in 5% (w/v) fat-free dry milk diluted in TBS for 1 h. Then, the membrane was washed 2 × 10 min in TBST (TBS with 0,1% (v/v) detergent Tween-20). For immunodetection, the membrane was incubated for 2 h in primary antibody (diluted in TBST) at room temperature. The following primary antibodies were used in the study: FLAG (Merck F1804), complex IV (COX1: Abcam 14705, Cambridge, UK, COX2: Abcam 110258, COX4i1: Abcam 14744, COX4i2: Abnova H00084701-M01, Taipei City, Taiwan, COX5a: Abcam 110262, COX6c: Abcam 110267), complex I (NDUFA9: Abcam, 14713), complex II (SDHA: Abcam 14715), complex III (Core 2: Abcam 14745), and citrate synthase (Abcam 129095). For quantitative detection, the corresponding infra-red fluorescent secondary antibodies (Alexa Fluor 680, Thermo Fisher Scientific; IRDye 800, LI-COR Biosciences, Lincoln, NE, USA) were used. Detection was performed using the fluorescence scanner Odyssey (LI-COR Biosciences) and signals were quantified by ImageLab software (Bio-Rad).

Proteins were isolated form exosomal fraction by adding SDS sample buffer and heating samples to 70°C for 10 min. Total of 7.5 μg of proteins was loaded in a single well and separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked for one hour at room temperature in Odyssey Blocking Buffer (LI-COR Biosciences), followed by incubation in primary antibodies diluted in Blocking Buffer with 0.1% Tween-20 overnight at 4°C. Membranes were rinsed with PBS followed by incubation with secondary antibody diluted in Blocking Buffer for one hour at RT. After final rinses, blots were scanned with Odyssey Infrared Imager (LI-COR Biosciences). Primary and secondary antibodies comprised: anti-CD63 (Santa Cruz Biotechnology, sc-15363; 1:1000), anti-Tsg101 (Genetex, gtx70255; 1:1000), goat anti-mouse Alexa Fluor 680 (Thermo Fisher Scientific, A-21058; 1:15,000), goat anti-rabbit Alexa Fluor 790 (Thermo Fisher Scientific, A-11369; 1:15,000).