Minichemi610

Cells were harvested and immediately lysed by RIPA strong lysis buffer (Beyotime, China) containing protease inhibitors and phosphatase inhibitors: 1% PMSF (Roche, Mannheim, Germany) and 2% PIC (Roche, Switzerland). After centrifugating for 20 min, protein concentration in the supernatant was collected in tubes and determined using a Pierce™ BCA protein assay kit (Thermo Fisher Scientific, USA). The supernatant was then mixed with protein loading buffer (NCM Biotech) and boiled at 100° C for 5 min. Equal amounts of proteins (10 μg) were electrophoresed and separated by SDS-PAGE gels (Bio-Rad, USA), transferred onto PVDF membranes (Immobilon®-P), and blocked with 5% BSA solution at room temperature. Then the membranes were incubated overnight at 4° C with indicated primary antibodies. Followed by washing membranes three times and incubating with secondary antibodies for 2 h at room temperature, and visualized by the NcmECL Ultra (A+B) chemiluminescence reagent (NCM Biotech) and exposed using a chemiluminescence imaging system (SAGECREATION MiniChemi 610), GAPDH was used as an internal control. Anti-rabbit TFAP2A antibody was purchased from Abclonal (A0416, 1:800), anti-rabbit PD-L1 antibody was purchased from Proteintech (66248-1-Ig, 1:3000), anti-mouse GAPDH antibody was purchased from Utibody (UM4002, 1:2000).

Cell lysates, different cell fraction extracts, and immune complexes were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), transferred to 0.2‐μm PVDF membranes, and incubated with different antibodies. After blocking with 3% (w/v) non‐fat dried milk, the membranes with targeted proteins were incubated with the corresponding primary antibodies overnight at 4°C. Membranes were then washed three times with PBST and incubated with HRP‐conjugated secondary antibody at room temperature for 30 min. After washing three times with PBST, the membranes were treated with the chemiluminescent substrates, and imaging was performed using the LAS‐4000 system (GE, CA, USA) or MiniChemi 610 (Sage Creation, Beijing, China).

Cells were lysed with RIPA buffer (Beyotime, China) containing a proteinase inhibitor cocktail (Keygen, China) and phosphatase (Beyotime). Protein sample concentrations were estimated using the BCA assay kit (Bio-Rad, USA). Protein samples were separated using a 10% SDS-PAGE gel and the separated proteins were subsequently transferred to a PVDF membrane (Millipore, USA). The membrane was blocked using 5% milk for 1 h at room temperature and was probed with primary antibodies at 4 °C overnight. Next, the membranes were incubated with the peroxidase-conjugated secondary antibody for 1 h at room temperature. GAPDH was used as the protein loading control. The blots were developed using the eECL Western Blot Kit (CWBIO Technology, China) and imaged using a chemiluminescence imaging system MiniChemi™ 610 with SageCapture^TM software (SAGECREATION, China). The antibodies used are listed in Supplementary Table 4.

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.

Western blotting was used to detect the cleavage promotion of HN proteins. BHK-21 cells were cotransfected with 1 µg each of HNs and F plasmid. After 24 h, the total protein was extracted from cells with 50 mM EDTA. The cells were pelleted, washed, and lysed. Then, the polypeptides were analysed by SDS–PAGE. An HN monoclonal antibody (1:3000) or HA-labelled specific mouse primary antibody (1:3000; Invitrogen, Carlsbad, CA, USA) and a secondary antibody (HRP-conjugated goat anti-mouse IgG, 1:3000; Invitrogen, Carlsbad, CA, USA) were used. The protein bands were exposed with a chemiluminescence imager (MiniChemi610; Sagecreation, Beijing, China). The protein load was normalized to the GAPDH signal (1:3000; Sungene Biotech, Tianjin Binhai New Area, China). The western blots were quantified by the F1/F0 densitometry ratio using ImageJ software.

Mice uterine tissues after cryogenic grinding or cell pellets were lysed in RIPA buffer (Cwbio, CW2333S) containing 1 mM PMSF (Sigma, 78830) on ice for 30 min before centrifuging at 4°C, 14 000 g for 15 min. The collected supernatants were quantified using BCA Protein Assay reagents (Pierce, 23227), then mixed with 5 × SDS loading buffer (Beyotime, p0015) and boiled for 10 minutes at 100°C. Proteins samples were separated by 10% SDS-PAGE and blotted onto nitrocellulose membranes (Pall, 66485). Membranes were blocked with 5% nonfat milk for 1 h at room temperature and then probed with the indicated primary antibodies for 1 h or overnight at 4°C. After washing thoroughly, the membranes were incubated with HRP-coupled secondary antibodies and visualized using Chemiluminescent Imaging System (Sagecreation, MiniChemi610). The images were analyzed with ImageJ software. Primary and secondary antibodies used for western blot were as follows: anti-CYP26A1 (1:1000, Abcam, ab151968), anti-Mannose Receptor (1:1000, Abcam, ab64693), anti-liver Arginase (1:1000, Abcam, ab60176), anti-GAPDH (1:1000, Cell signaling technology, 2118), HRP-conjugated goat anti-rabbit IgG (1:10000, Thermo, 31460) and HRP-conjugated bovine anti-goat IgG (1:5000, Jackson ImmunoResearch Laboratories, 805-035-180).