Pvdf membrane

Total proteins were extracted according to the whole protein extraction kit (Solarbio, Beijing, China) and protein concentrations were measured using the BCA protein assay kit (Solarbio, Beijing, China). Next, total proteins were separated by SDS/PAGE electrophoresis technique and then transferred to PVDF membrane (EpiZyme, Shanghai, China), closed at room temperature for 1 h. Primary antibody was incubated overnight at 4°C, then goat anti-rabbit IgG (Beyotime, Shanghai, China) was treated at room temperature for 1 h. Finally, the proteins were developed using a developing solution (Thermo Fisher Scientific, America). The following primary antibodies were used: CST1 (1:1000,CST, America), MEK1/2 (1:1000, CST, America), p-MEK1/2(Ser217/221)(1:1000, CST, America), p44/42 MAPK (Erk1/2) (1:1000, CST, America), p-p44/42 MAPK (Erk1/2) (1:1000, CST, America), CREB (1:1000, Affinity Biosciences, America), p-CREB(ser133)(1:1000, Affinity Biosciences, America), ELK1(1:1000, Affinity Biosciences, America), p-ELK1(ser383)(1:1000, Affinity Biosciences, America), E-cadherin (1:1000, CST, America), MMP2 (1:1000, CST, America), Slug (1:1000, CST, America), and β-actin (1:1000, Beyotime, Shanghai, China).

Skin tissue was homogenized in RIPA (Radio Immuno Precipitation Assay) lysis buffer, protease inhibitor, and phosphatase inhibitor (Boster, Wuhan, China) to extract the total protein. The protein concentration was determined using BCA protein assay kit. The protein was denatured in gel sample buffer at 100 °C for 5 min, separated by 10% SDS-PAGE, transferred to a polyvinylidene fluoride (PVDF) membrane (EpiZyme, Shanghai, China), and blocked with 5% (w/v) non-fat dry milk in TBST buffer (Tris-Buffered Saline with Tween-20). The membrane was incubated with the primary antibody against NF-κВ (BA1297-2, Boster, Wuhan, China), TNF-α (BA14901, Boster), IL-6 (BA4339-2, Boster), COX-2 (BA3708, Boster), ERK (GB112238, Servicebio, Wuhan, China), JNK (M02608-3, Boster), P38 (A00176-2, Boster), AP-1 (GB11270, Servicebio), MMP-1 (A00733-1, Boster), and MMP-2 (A00286, Boster) at 4 °C overnight, followed by washing at TBST, and then incubated with goat anti-rabbit HRP (horseradish peroxidase)-IgG (BA1055, Boster) as the secondary antibody for 45 min. After washed with TBST 3 times for 10 min, membrane was incubated in SuperSignal ECL Western substrate (Biosharp, Hefei, China). The protein band was visualized using the Chemiluminescent Imaging System (Tanon-5200SF, Shanghai, China). The intensity was quantified by Image J version 1.8.0 program.

Based on our previous study (Duan et al., 2022 (link)), NTS tissues were punched as protocol in quantitative polymerase chain reaction (qPCR). The tissue of NTS was lysed with lysate (Beyotime, China). The protein concentration of NTS sample was measured by BCA Protein Assay Kit (Beyotime, China), diluted with 5× SDS-PAGE uploading buffer (Solarbio, China) and PBS (Solarbio, China) to denature by boiling. Proteins were separated by SDS-PAGE in the 10% gradient gel (EpiZyme, China) and transferred to a PVDF membrane (Sigma, United States). After transfer, the PVDF membrane was incubated with 5% skimmed milk (EpiZyme, China) in TBST for 1.5 h at room temperature. The primary antibody (anti-AT1R, No. ab124734, Abcam) was diluted at 1:1000 in TBST, and then the PVDF membrane was incubated overnight at 4°C. The PVDF membrane was washed 3 times and then incubated with the secondary antibody for 2 h at room temperature. And the immunostaining bands were detected by an automatic chemiluminescence image analysis system (Tanon Science & Technology, China).

Western blot was carried out essentially as described^{30 (link)} with slight modifications. Total protein was extracted using a whole protein extraction kit (Solarbio, Beijing, China), and protein concentrations were measured using a BCA protein assay kit (Solarbio, Beijing, China). Next, total proteins were separated by SDS/PAGE electrophoresis and then transferred to a PVDF membrane (EpiZyme, Shanghai, China) at 25 °C for 1 h. Overnight incubation with the primary antibody at 4 °C was performed, and then goat anti-rabbit IgG (Beyotime, Shanghai, China) was added at 25 °C for 1 h. Finally, the proteins were detected using a developing solution (Thermo Fisher Scientific, USA). The following primary antibodies were used: CST1 (1:1000; ab307416, Abcam, British), MEK1/2 (1:1000; #9122, CST, USA), p-MEK1/2 (Ser217/221) (1:1000; #9121, CST, USA), p44/42 MAPK (Erk1/2) (1:1000; #9102, CST, USA), p-p44/42 MAPK (Erk1/2) (1:1000; #9101, CST, USA), GRIM19 (1:1000; A18071, Abclonal, China), SDHA (1:1000; A16204, Abclonal, China), UQCRC2 (1:1000; A4184, Abclonal, China), COX IV (1:1000; A6564, Abclonal, China), ATP5A1 (1:1000; A11217, Abclonal, China), VDAC1 (1:1000; A19707, Abclonal, China), MMP2 (1:1000; #4022, CST, USA), and β-actin (1:1000; AF5003, Beyotime, Shanghai, China). All experiments were repeated three times.

Cells and tissue proteins were lysed using sodium dodecyl sulfate (SDS) lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with PMSF (Solarbio, Beijing, China) and Phosphatase Inhibitor Cocktail (Beyotime Biotechnology, Shanghai, China). Proteins were separated using SDS polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes (Epizyme, China). Next, the membranes were cut horizontally, incubated with primary and secondary antibodies. The protein bands were detected using ECL reagent (Cytiva, USA). Images were captured using a Bio-Rad Multifunctional chemiluminescence imaging system. GAPDH or β-tubulin was used as a loading control. Antibody information is shown in Additional file 1: Table S1.

The protein extraction kit (KeyGEN, China) extracted proteins from the EXOs or cells. Then, a BCA assay kit (Thermo Fisher Scientific, USA) detected the concentrations of proteins. The antibody of β-actin (#3700, 1:5,000, CST, USA) was used as a loading control. About 15 μg of protein was separated by 10% SDS-PAGE and transferred to PVDF membranes (EpiZyme, China). Next, the membranes blocked the non-specific antigen in 5% BSA for 1 h at room temperature. After that, the membranes were incubated with specific antibodies at 4°C for 12 h. Then, the membranes were washed with tris buffered saline with Tween-20 three times, and followed by incubation with secondary antibodies (1:5,000, Abcam, USA) for 2 h. After washing, the bands were detected using Gel-Pro Analyzer software (Media Cybernetics, USA).