Ab151968

Mice uterine proteins were extracted using lysis buffer (Applygen, Beijing, China) with protease inhibitors (Roche). The concentration was determined by following the specifications of the Bicinchoninic Acid Protein Assay Kit (Pierce, Rockford, IL). Proteins were separated by 10% SDS‐PAGE and electrical transferred onto a nitrocellulose membrane (Pall, New York, NY). After being blocked in 5% skimmed milk powder made from TBST solution at room temperature for 3‐4 hours, the membranes were washed with TBST solution and incubated with primary antibodies at 4°C overnight. Rabbit source polyclonal anti‐CYP26A1 antibody (1:1000; ab151968, Abcam), anti‐Id2 antibody (1:500; PA5‐49683, invitrogen) and anti‐GAPDH antibody (1:1000; mAb #5174, CST) were used. Then the membrane were washed thoroughly in TBST solution and incubated with goat antirabbit IgG conjugated with horseradish peroxidase (HRP; KPL, Gaithersburg, MD) diluted by 1:10,000 in TBST at 25°C for 1 hour. Finally, the membrane was visualized and quantified on a Gene Gnome XRQ Chemiluminescence detector (Syngene, Cambridge, UK) using ECL Detection Kit (Pierce, Rockford, IL) and GeneSys software (VilberLourmat, France). The relative protein levels were analysed by Bio‐Rad Quantity One software (Bio‐Rad, Hercules, CA) and standardized to GAPDH.

Mouse uterine tissue was ground into powder in liquid nitrogen and added to RIPA Lysis Buffer (CW2334S, Cwbio) containing 1 mM PMSF (78830, Sigma). Total protein was extracted following the instructions of the RIPA Lysis Buffer kit. The Bicinchoninic Acid Protein Assay Kit (23227, Pierce) was used to detect the protein concentration. Proteins were separated using 10% SDS‐PAGE and then transferred from the gel onto a nitrocellulose membrane (66485, Pall). The membrane was blocked with 5% skimmed milk and then incubated overnight with primary antibodies at 4°C. After washing thoroughly with TBST solution, the membranes were incubated with HRP‐coupled secondary antibodies at room temperature for 1 h and then visualized using a chemiluminescence imaging system (MiniChemi 610, Sagecreation). The data were analysed using ImageJ software. The primary and secondary antibodies used for Western blotting included anti‐CYP26A1 (ab151968, 1:1000, Abcam), anti‐GAPDH (2118, 1:1000, Cell Signaling Technology) and goat anti‐rabbit IgG (H + L) HRP (31460, 1:10,000, Thermo Fisher).

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).