β tubulin

Protein concentration was quantified by the BCA method (CW Biotech, Beijing, China) and 30 µg of protein extracts from left lung tissue was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8%). After electrophoresis, proteins were transferred to an NC membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. The membranes were probed via overnight incubation at 4°C with each of mouse or rabbit antibodies raised against: (1) Rcn3 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA); (2) GRP78 (1:2000, Santa Cruz Biotechnology, Dallas, TX, USA); (3) Cleaved caspase-3 (1:1000, Cell Signaling Technology, USA); (4) β-actin (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA); (5) β-Tubulin (1:5000, Yeasen, Shanghai, China); (6) GAPDH (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA); at 4°C overnight. After washing with TBST, secondary antibodies (Zhongshan Golden Bridge, Beijing, China) were applied at room temperature for 2 hours. Protein bands were visualized using ECL reagent. Image J software was used for subsequent quantification and statistical analysis.

Experiments were performed as described (42 (link)). Briefly, cells were lysed in lysis buffer and cell lysate containing 30 μg protein was used for Western blots. Membranes were incubated with the primary antibody against EOMES (Cat No. ab23345, Abcam, 1:1000), FAM46B (Cat No. 23149-1-AP, Proteintech, 1:1000), GAPDH (Cat No. sc-47724, Santa Cruz Biotechnology, 1:5000), β-Tubulin (Cat No. 30303ES50, YEASEN, 1:1000) and Laimin B1 (Cat No. 16048, Abcam, 1:1000). The membranes were then incubated with IRDye 680LT Donkey anti-Rabbit IgG (Cat No. 926-68023, Li-COR Biosciences) or IRDye 800CW Donkey anti-Mouse IgG (Cat No. 926-32212, Li-COR Biosciences) as secondary antibodies and visualized on an Odyssey Infrared Imager (Li-COR Biosciences).

Radioimmunoprecipitation assay lysis buffer (Ther- mo Fisher Scientific) was used to obtain protein extracts from human placentas and cell cultures. The concentration of the obtained protein sample was measured with a BCA protein determination kit (Thermo Fisher Scientific). The protein samples were separated by SDS-PAGE and then transferred to PVDF membranes. Five percent skimmed milk was used to block the membranes at room temperature for 1 hour, and then the membranes were incubated with antibodies (BST2 and MMP2 (Abcam, UK), GAPDH, β-actin and β-tubulin (Yeasen, China) at 4°C overnight. Corresponding horseradish peroxidase-conjugated secondary antibodies were added for incubation at room temperature for 1 hour. The color was developed by chemiluminescence (Bio-Rad, Germany), and the imaging results were analyzed with a gel imaging system [25 (link)].