Cryab

Proteins isolated from pig endometrium tissue (extraction steps described above) were used to validate the iTRAQ results. 30 μg of protein was separated by SDS-PAGE and then electro-transferred onto PVDF membrane (Millipore). Membranes were blocked overnight with blocking reagent at 4 °C and then incubated with one of five primary antibodies; CTSB, GLA, CRYAB, DPP4, or ASAH1 (13,000, Abcam) for 2 h at room temperature. Membranes were rinsed six times in TBST (20 mM Tris–Cl, 140 mM NaCl, pH 7.5, 0.05% Tween-20) for 30 min, and then incubated with a secondary antibody (goat-anti rabbit IgG HRP-conjugate, 1:8000, Abmart) for 2 h at room temperature. Membranes were washed again with TBST for 30 min. The membranes of Western blot were incubated with ECL chemiluminescent substrate (ThermoFisher, USA) for 5 min at darkroom. The light output of ECL can be captured using film (Koda, China). Films were imaged with scanner and Image J software (https://imagej.nih.gov/ij/) was used to compare the density of bands. Results are presented as means ±SEM. Differences were tested for statistical significance using ANOVA. p < 0.05 was considered the threshold for statistical significance (*, P < 0.05; **, P < 0.01).

Immunofluorescence was performed as described previously^{27 (link)} with primary antibodies against F4/80 (Santa Cruz), CD206 (BioLegend), E-cadherin (Proteintech, Wuhan, China), N-cadherin (Proteintech, Wuhan, China), Slug (Proteintech, Wuhan, China) and CRYAB (Abcam).