Phenyl methyl sulfonyl fluoride (pmsf)

Macrophages were harvested, and proteins were extracted using RIPA buffer (Beyotime) containing protease inhibitors PMSF (Servicebio, China) and cocktail (Roche). For the protein preparation of the culture supernatants, 700 μL culture supernatants were collected and mixed with 700 μL 100% methanol and 175 μL trichloromethane. Supernatants were centrifuged at 13,000 rpm for 5 min at 4 °C to precipitate the proteins. The remaining proteins were resuspended in 20 μL10% SDS. 30 µg of cell lysate samples or culture supernatant samples were resolved by a 12% SDS/PAGE. Western blotting was performed as described previously [34 (link)]. The blots were visualized by ChemiDoc XRS (Bio-Rad, USA). The relative band intensity was quantified using the Image Lab Analyzer software (Bio-Rad). α-tubulin was used as the loading control. The antibodies used in the study are shown in Table 1.

Cells lysates were prepared using RIPA buffer (Beyotime, China) containing PMSF and phosphatase inhibitor (Servicebio, China). The extracts were clarified by centrifugation at 12,000 rpm for 10 min at 4°C and total protein concentrations estimated using the BCA Protein Assay Kit (Beyotime, China). Equal protein amounts were prepared in SDS-PAGE loading buffer and heated at 100°C for 5 min before protein separation by SDS PAGE and transfer to PVDF membranes (Millipore). After blocking with skim milk solution for 90 min, membranes were incubated with the indicated primary antibodies. (Src rabbit monoclonal antibody, 1:1000, CST; Phospho-Src Family (Tyr416) (D49G4) rabbit monoclonal antibody, 1; 1000, CST; t-FUNDC1 rabbit polyclonal antibody, 1:500, abcepta; p-FUNDC1 (Tyr18) rabbit polyclonal antibody, 1:500, abcepta; LC3 rabbit polyclonal antibody, 1:1000, Proteintech; P62 rabbit polyclonal antibody, 1:1000, CST; GAPDH Mouse monoclonal antibody, 1:3000, Antgene, China; Detailed antibody information is provided in Supplementary Table S1) followed by the appropriate anti-rabbit or mouse HRP-conjugated secondary antibodies. The membranes were incubated with ECL solution and protein bands visualized in an imaging system. The intensities of bands were quantified using Image J software. Uncropped Western blot images are provided in Supplementary Figure S1.

Protein samples were isolated from tissues or cells using RIPA lysis buffer (Servicebio, Wuhan, China) containing 1% protease and phosphatase inhibitors (PMSF; Servicebio). A BCA protein assay kit (Biyuntian Biotechnology) was used for protein quantification. Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis was applied to separate proteins of different molecular weights, and proteins were transferred to a polyvinylidene fluoride (Servicebio) membrane. The membrane was blocked with 5% skim milk for 90 min at room temperature and subsequently incubated with primary antibodies (Proteintech; IL-1β 1:1000; MMP9 1:1000; β-actin 1:25,000) at 4 °C overnight, followed by incubation with HRP-conjugated secondary antibodies for 2 h at room temperature.

Total protein was extracted from cells using a RIPA buffer (Solarbio) supplemented with PMSF (Servicebio) (100:1). Protein was separated on 10% SDS-PAGE gels. The proteins were transferred to PVDF membranesm, and then blocked with 5% non-fat milk for 2 h. The membranes were washed with PBST three times (5 min/time) and incubated with the primary antibodies (Abcam) at 4°C for overnight. Then the membranes were washed three times using PBST and incubated with secondary antibody conjugated with HRP (Abcam) for 1 h at room temperature. Signals were detected by ECL Plus (Solarbio). β-Actin was used as an internal control.

Proteins were first extracted by adding RIPA lysate and protease inhibitor PMSF(Servicebio, Wuhan, China), denatured with SDS for 10 min using 15% of the separation gel and concentrated gel were run in electrophoresis solution at a constant voltage of 80V–120 V for 80 min. Membranes were rotated at a constant current of 260 mA for 90 min. Then incubation were performed in skimmed milk at 37°C for 2 h. Membranes were incubated overnight at 4°C with rabbit anti-TXNDC12 (Abclonal, Wuhan, China, 1:1,000), followed by incubation with the secondary antibody (Bioworld, United States, 1:10000) at 37°C for 60 min.

Total protein was extracted from endometrium tissues using RIPA buffer (Servicebio, China) supplemented with 1% PMSF (Servicebio, China). Protein quantification was performed using an Enhanced BCA Protein Assay Kit (Servicebio, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out for protein separation. Then, the protein samples were transferred onto the PVDF membrane (Servicebio, China) and blocked in 5% BSA (Servicebio, China). Subsequently, the membranes were incubated with the primary antibodies against HOXA10 (ab191470, Abcam, UK), LIF (ab138002), VEGF (GB11034B, Servicebio, China), VEGFR2 (GB11190, Servicebio, China), P-PI3K (GB11190, Servicebio, China), AKT (GB111114, Servicebio, China), P-AKT (AF3242, Affinity, USA), and ACTIN (GB15001, Servicebio, China) at 4°C overnight. After washing with TBS-T three times, the membranes were then incubated with the secondary antibody at RT for 1h and detected using an ECL plus kit (G2019, Servicebio, China). PhotoShop software (alphaEaseFC, Alpha Innotech, USA) was used to remove the color, and Alpha software (Adobe PhotoShop, Adobe, USA) was used to analyze the optical density of the target band.