Ecl luminescent solution

The total protein was extracted and redistributed by 10% SDS–PAGE after being mixed with 5× loading buffer. The mixture was separated by using an electrophoresis program, according to molecular weight, and transferred to 0.22 μm PVDF (Millipore, MA, USA) membrane by JY–ZY5 transfer electrophoresis tank (Junyi, Beijing, China) at 150 mA for 1.5 h. After 1 h of blocking with 5% skimmed milk (BD Biosciences, NJ, USA), the protein of the stem cells was bound by Tuj1 antibody (Beyotime, Shanghai, China), GFAP antibody (Beyotime, Shanghai, China), and Nestin antibody (Beyotime, Shanghai, China) at 4 °C for 16–20 h. The secondary antibody with HRP (Proteintech, IL, USA) was used to incubate at 37 °C for 1 h, and the protein abundance was observed after mixing with ECL luminescent solution (Beyotime, Shanghai, China).

About 50 mg of lamprey tissue was homogenized in RIPA Lysis Buffer (Beyotime, Shanghai, China) plus protease inhibitors (Beyotime) in an ice bath and then centrifuged. The supernatant was added to the loading buffer and boiled for 8 min to prepare a protein sample. The protein samples were first separated by SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF, Millipore) membrane for 75 min, and sealed with 5% skimmed milk powder for 3 h. Then the Lja-BCAP antibody (1:1000 v/v) was applied and incubated at 4 °C. After at least 4 h, the membrane was washed with 1 × TBST buffer (Tris buffer plus 0.05% Tween-20) for 10 min each time, five times in total. The secondary antibody (Goat anti-rabbit, Beyotime) was added at a ratio of 1:5000 and incubated at 37 °C for 1 h. the membrane was washed with 1 × TBST buffer, 10 min each time, four times in total. Finally, ECL luminescent solution (Beyotime) was added, and FluorChem Q multicolor fluorescence chemiluminescence imaging system (ProteinSimple, San Jose, CA, USA) was used to detect the fluorescence signal. The β-Actin Mouse mAb (ABclonal) was used to identify lamprey β-actin as internal control [32 (link)]. Densitometry data generated for WB to compare protein concentrations were analyzed with the software ImagePro 6.0.

Total protein was extracted by lysing L02 cells with radioimmunoprecipitation assay buffer (Solarbio) containing an inhibitor cocktail (Roche), and the protein concentration was measured using a BCA kit (Beyotime). Equal amounts of protein (20 μg) were separated using 12% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). Membranes were blocked with a blocking solution containing 5% milk. After 1 h, the membranes were incubated overnight at 4°C with primary antibodies against TLR4 (cat. no. 14358; Cell Signaling Technology) and p65 (cat. no. 8242; Cell Signaling Technology) and nuclear p‐p65 (cat. no. 3033; Cell Signaling Technology) and Bax (cat. no. 14796; Cell Signaling Technology) and Bcl‐2 (cat. no. 4223; Cell Signaling Technology). The next day, membranes were washed with TBST. The membranes were then incubated with HRP‐labeled secondary antibodies for 2 h and developed with ECL luminescent solution (Beyotime).

The HUVECs in each group were treated based on the experimental purpose, the cell plate was removed, and the cells washed with cold PBS solution. Experimental procedures were performed strictly according to the manufacturer's instructions. Total cell protein was extracted from RIPA lysate and the protein concentration was determined using the BCA method. Equal amounts of protein samples from each group were separated using SDS-PAGE electrophoresis and transferred to PVDF membranes (Bio-Rad). All blots were excised prior to hybridization with antibodies. PVDF membranes were blocked with 50 g/L skim milk followed by incubation overnight at 4 °C with primary antibodies: Cleaved Caspase-3 (1:1000, Cell Signaling Technology, Danvers, MA), Cleaved PARP (1:1000, Beyotime), anti-HMGB1 (1:1000, Abcam, Waltham, MA, USA), anti-Akt (1:1000, Beyotime), anti-phosphorylated Akt (p-Akt, 1:1000, Beyotime), anti-NF-κB p65 (1:1000, Boster, Wuhan, China), and GAPDH (1:1000, Beyotime). The next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Boster) for 1 h at 37 °C. Then, ECL luminescent solution (Beyotime) was added to expose and develop labeled antibodies. The relative expression levels of HMGB1, Akt, and p-Akt proteins were analyzed using Image J software. The experiment was repeated three times.

Cells, exosomes, and cell lysates were lysed on ice with RIPA lysis buffer (Solarbio, China). After the total protein was extracted, sonication was performed in an ice bath, and centrifugation was conducted at 10,000 rpm at 4°C for 20 min. Then, the protein supernatant was collected. A BCA kit (Solarbio, China) was used to check the protein concentration. Further, 20 μg of total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% nonfat milk blocking solution at ambient temperature for 1–2 h, diluted primary antibodies (anti-HSP70, anti-CD63, anti-TRIM67, and anti-β-actin, CST, USA) were added for incubation overnight at 4°C. After washing the membranes twice with phosphate-buffered saline with tween (PBST), a diluted enzyme-labeled secondary antibody (Zhongshan Golden Bridge, China) was added for another 1-h incubation at ambient temperature. After washing with PBST for another three times, electrochemiluminescence (ECL) luminescent solution was added (Beyotime, China) for following exposure and photography in a gel imager. Image-pro plus software was used to analyze the gray value of the protein bands, and β-actin served as an internal control for the analysis of the relative expression level of the target protein.