Sa00001 1 2

The total protein was extracted from HOKs treated with different concentrations of arecoline using SDS lysis buffer (P0013G; Beyotime). The total protein concentration was detected using Pierce™ BCA Protein Assay Kit (23225; Thermo Fisher Scientific). A total of 40 µg protein was added to 10% polyacrylamide gel and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% skim milk for 1 h at room temperature. After PBST washing three times, the membranes were incubated with primary antibodies overnight at 4° overnight: p16 (1:2000, ab108349, Abcam), p21 (1:2000, 2946; Cell Signaling Technology), p53 (1:1000, 48818; Cell Signaling Technology), transforming growth factorβ1 (TGF-β1) (1:1000, ab215715; Abcam), and GAPDH (1:1000, 5174T, Cell Signaling Technology). Then, the membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibodies (anti-Rabbit/mouse 1:5000∼10000, SA00001-1/2; Proteintech) for 1 h at room temperature. The membranes were developed using SuperSignal™ West Pico PLUS chemiluminescent substrate (34580; Thermo Fisher Scientific) and detected by a chemiluminescent imaging system (MiniChemi 610; Sage Creation, Beijing, China). and analyzed using the ImageJ 5.0 version software.

For total protein extraction, the cells were washed with PBS, lysed in lysis buffer (87787, Thermo Fisher Scientific, U.S.A.) following the addition of a protease inhibitor, incubated on ice for 5 min, and centrifuged at 13000× g for 10 min at 4°C. Protein concentration was determined with a bicinchoninic acid assay (BCA) kit (P0012, Beyotime). We separated proteins using 10% SDS/PAGE and transferred them to PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h at room temperature and incubated with primary antibodies against RIP3 (1:1000; ab62344, Abcam), pRIP3 (1:1000; 93654, CST), MLKL (1:1000; ab184718, Abcam), pMLKL (1:1000; 91689, CST), peroxisome proliferator-activated receptor γ (PPARγ; 1:1000; ab178860, Abcam), vascular endothelial growth factor-A (VEGFA; 1:1000; 19003-1-AP, Proteintech), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:50000; 60004-1, Proteintech) as an internal control at 4°C overnight. After being extensively washed with Tris-buffered saline with Tween 20 (TBST), the membranes were incubated with anti-rabbit/mouse secondary antibodies (1:10000; SA00001-1/2, Proteintech) for 1 h at room temperature. Protein was developed with ECL reagent (Millipore; Sigma–Aldrich, Germany) and visualized using a ChemiDoc MP Imager (Bio-Rad Laboratories, U.S.A.). The density of bands was determined by ImageJ (version 1.53c).

RIPA lysate (Solarbio) and protease inhibitor PMSF (Solarbio) with a final concentration of 1 mM were added into the cells, and then, the total protein of the cells was extracted by centrifugation. The total protein concentration was tested by the BCA method, the final loading protein concentration was adjusted to 5–10 μg/μl, the sample was loaded, and 10% SDS-PAGE separation gel was applied for gel electrophoresis. The target protein was transferred to PVDF membrane by the semiwet transfer method, sealed with 5% skimmed milk powder for 1 h, and then added with SIRT1 (60303-1-Ig, Proteintech, 1 : 4000) and MMP9 (10375-2-AP, Proteintech, 1 : 1000) primary antibody diluent at 4°C overnight. After being washed with 0.1% PBST buffer for 3 times, the corresponding HRP-labeled secondary antibody (SA00001-1/2, Proteintech, 1 : 5000) was added and incubated at room temperature for 1 h and then washed with 0.1% PBST buffer for 3 times. ECL chemiluminescence solution (Solarbio) was evenly dripped on PVDF membrane, and the expression of protein was analyzed by ImageJ.

The proper amount of lysate (R0020, Solarbio) was added to the cells of the control and treatment groups to extract the total proteins by centrifugation after cell lysis. The BCA kit (P0012S, Biyuntian) was used to detect the protein concentration. The total proteins were gelled in an SDS-PAGE gel, and the target protein was transferred to the PVDF membrane by the semiwet transfer method. TLR3 (1 : 2000, ab62566, Abcam), NF-kBP65 (1 : 2000, ab16502, Abcam), and GAPDH (1 : 5000, ab8245, Abcam) antibody dilutions were added after BSA closure, which was kept at 4°C overnight. Then, it was washed with 0.1% PBST 3 times. The corresponding sheep anti-rabbit/mouse immune second antibody (1 : 2000, SA00001-1/2, Proteintech) was added to incubate for 1 h at room temperature. ECL chemiluminescence solution (P0018FS, Biyuntian) was added after washing 3 times. The image was developed, fixed, and recorded in a dark room, and the processing software ImageJ 1.52 was used for analysis.
http://www.ptgcn.com/Products/HRP-conjugated-Affinipure-Goat-Anti-Mouse-IgG-H-L--secondary-antibody.htm.