Two color near infrared laser imaging system

SLC4A1-EC
protein (8 μg) was coincubated with peptides XRB2, XRE4, and
XRH7 (4 μg) at 37 °C for 1 h, and peptides XRB2, XRE4,
and XRH7 alone and SLC4A1-EC protein alone were used as controls.
After the reaction, glycerol (final concentration 10%) was added,
and the peptide–protein mixture was heated at 99 °C for
15 min for denaturation and then subjected to SDS–PAGE. After
electrophoresis, fluorescent protein imaging was first performed on
a two-color near-infrared laser imaging system (LI-COR, USA). Then,
Coomassie brilliant blue staining was performed for further observation
and verification.

After the RBCs, RBC-EVs,
MSCs, and MSC-EVs
were lysed with protein RIPA lysis buffer (Beyotime, China), an aBCA
protein detection kit (Pierce, Rockford IL, USA) was used to measure
the total protein content. The total mass of the cells and EV proteins
was 75 μg, and the proteins were separated by SDS–polyacrylamide
gel electrophoresis (SDS–PAGE) using a 4% concentration gel
and a 10% separation gel. After electrophoresis, the proteins were
transferred to PVDF membranes. The membranes were sealed with 5% skim
milk powder in Tris-buffered saline containing 0.1% Tween 20 for 1.5
h and then incubated with primary antibodies against SCL4A1, CD63,
HSP70, flotillin, CD9, CD81, and calnexin (CANX) (Abcam, England)
overnight at 4 °C. The next day, the protein membrane was washed
and incubated with a secondary antibody at room temperature for 1.5
h, and the protein bands were detected by a chemiluminescence kit.
Protein imaging was performed using a two-color near-infrared laser
imaging system (LI-COR, USA).