Anti mmp2

Protein extraction and blotting was performed as described previously [7 ]. Western blots were probed with the following antibodies anti-BMP5 (Abcam, ab88064, 1:500), anti-PCNA (Immunoway, 1:5000), anti-MMP2 (Proteintech, 1:500), anti-MMP9 (Proteintech, 1:1000), anti-E-cadherin (BD Biosciences, 1:1000), anti-EPSTI1 (Proteintech, 1:500) and anti-GAPDH (Immunoway, 1:5000).

Western blot assay was performed as the previous study described [48 (link)]. Total proteins were extracted using RIPA buffer (#FD011, Hangzhou Fude Biological Technology) and quantified by BCA protein assay kit (#P0011, Beyotime). In total, 1 μg of proteins were separated by 10% SDS-PAGE and transferred to PVDF membrane (#IPVH00010, Merck Millipore). After blocked with 5% Non-Fat Milk and incubated with specific antibody at 4 °C overnight, followed by HRP-conjugated secondary antibody incubation, the membrane was imaged with imager (Biorad ChemiDoc MP, Biorad). The antibodies used in this study were listed: anti-GAPDH (#5714, Cell Signaling Technology), anti-METTL1 (#ab157097, Abcam), anti-E-cadherin (#20874-1-AP, Proteintech), anti-N-cadherin (#66219-1-AP, Proteintech), anti-VIMENTIN (#10366-1-AP, Proteintech), anti-MMP2 (#10373-2-AP, Proteintech), anti-CDK4 (#11026-1-AP, Proteintech), anti-P16 (#18769, Cell Signaling Technology), anti-ATF3 (#ab254268, Abcam).

Total proteins were extracted by the Cell Lysis Buffer (Beyotime, China). The protein quantification from the total extracted cell protein lysates was detected by the BCA protein assay, then the same 10 μg of protein samples were separated by the SDS-PAGE and transferred onto PVDF membranes. The proteins on the membrane were then blocked with 5% skim milk for 30 min and incubated overnight at 4 °C with anti-E-Cadherin (#20874-1-AP, Proteintech, China, 1:5000), anti-MMP2 (#10373-2-AP, Proteintech, China, 1:500), anti-ZO-1 (#13663, Cell Signaling Technology, USA, 1:1000), and anti-GAPDH (#10494-1-AP, Proteintech, China, 1:10,000) antibodies. The membranes were incubated with the corresponding secondary antibody (#SA00001-1, #SA00001-2, Proteintech, China, 1:5000) at room temperature for 30 min. Last, membranes were incubated with chemiluminescent substrate according to the manufacture’s protocol (#E412, Vazyme, China) and visualized using Tanon-4600 automatic chemiluminescence/fluorescence image analysis system (Shanghai, China). The experiments were repeated twice.

Cells were lysed with RIPA lysate (Beyotime, Shanghai, China), and the concentration of protein was determined by the BCA assay kit (Solarbio, Beijing, China). Briefly, the equal amount of protein was separated with 10% SDS-PAGE (Epizyme, Shanghai, China), and transferred to PVDF membranes (Millipore, MA, USA). The PVDF membranes were blocked with 5% skim milk and incubated at 4 °C overnight with primary antibody, including anti-Fibronectin (Cell Signaling Technology, MA, USA), anti-N-cadherin (CST), anti-E-cadherin (CST), anti-MMP2 (CST), anti-MMP9 (CST), anti-Cytokeratin (Proteintech, Wuhan, China), anti-Vimentin (CST), anti-Ran (Proteintech), anti-Akt (CST), anti-p-Akt (CST), GSK3β (CST), anti-p-GSK3β (CST), anti-β-catenin (CST). β-actin (CST) was used as internal controls. Then the PVDF membranes were incubated with a secondary antibody for 1 h. The blots were scanned by Amersham Imager 600 (GE, Boston, MA, USA). Each sample was repeated three times and the three independent western blot bands were quantitatively analyzed by Image J software (NIH, Bethesda, Maryland, USA).

The procedure of western blot was the same as the previous paper [23 (link)]. In brief, the total protein from the cells was lysed with radio immunoprecipitation assay buffer (Solarbio Biotech). Protein concentrations were measured using BCA protein assay kit (Beyotime) according to the manufacturer’s instructions. Then, equivalent amount of protein were separated by SDS-PAGE and transferred into polyvinylidene fluoride membranes (Bio-Rad). After blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight and then incubated with secondary antibodies for 2 h at room temperature. Bands were visualized with an ECL detection kit (Thermofisher) using ChemiDoc XRS Plus (BioRad) and analyzed using the Image J software. All primary antibodies and secondary antibodies in the study are listed as follows: anti-ACSM5 antibody (1:500, Proteintech), anti-collagen I (1:1000, Abcam), anti-collagen III (1:1000, Abcam), anti-MMP2 (1:1000, Proteintech), anti-MMP13 (1:1000, Proteintech), and anti-β-actin antibody (1:5000, Abcam).

The embedded placental tissue sections were treated using standard immunohistochemical techniques. Antibodies used in immunohistochemistry experiments included an anti-PD-L1 Ab (1:800 dilution; Proteintech, 66,248–1-Ig), anti- p-ERK Ab (1:300 dilution; CST, Thr202/Tyr204, mAb #4370), anti-MMP-2 (1:200 dilution; Proteintech, 10,373–2-AP) and anti- MMP-9 Ab (1:200 dilution; Proteintech, 10,375–2-AP). Immunoreactivity was evaluated independently by two investigators who were blinded to experimental protocol according to the intensity and extent of staining. Immunohistochemistry images were obtained with confocal microscopy (Leica DM4000B) and immunoreactivity using at least 3 random microscopes field of view. The experimental results were analyzed using Image-Pro Plus software (version 6.0; Media Cybernetics).

The antibodies were as follows: anti-Rab32 (Proteintech, #10999-1-AP, 1:1000), anti-MMP2 (Proteintech, #10373-2-AP, 1:1000), anti-MMP9 (Proteintech, #10375-2-AP, 1:1000), anti-β-Actin (CST, #4970, 1:1000), anti-N-cadherin (CST, #13116, 1:1000), anti-E-cadherin (CST, #3195, 1:800), anti-Vimentin (CST, #5741, 1:1000), anti-p-Drp1(Ser616) (CST, #3455, 1:1000), anti-p-Drp1(Ser637) (Abmart, #TD2980, 1:1000), anti-Drp1(CST, #8570, 1:1000), anti-Tom20 (Abcam, #ab56783, 1:1000), anti-p-ERK_1/2 (CST, #4370, 1:2000), anti-ERK_1/2 (CST, #4695, 1:2000). All secondary antibodies (HRP-conjugated anti-rabbit and anti-mouse, #A0208 and #A0216, 1:2000) were purchased from Beyotime Biotechnology (Shanghai, China). Anti-HA-tag (CST, #3724, 1:50 for IP and 1:1000 for western blot) and anti-IgG antibody (Servicebio, #GB23301, 1:100 for IP). Mdivi-1 (#S-7162), ERK_1/2 inhibitor SCH772984 (#S-7101) were purchased from Selleck.