Optical luminometer

Total protein of PE embryonic tissues, EVs or transfected cells was extracted, and the bicinchoninic acid protein quantitative kit (ThermoFisher Scientific) was employed to determine protein concentration. Protein was electrophoretic separated by 10% sodium dodecyl sulphate polyacrylamide gels and transferred to nitrocellulose membranes (ZeYE Biological). The membrane was blocked with 5% skim milk powder and then placed in a 3% BSA blocking buffer. Then, membrane was probed with primary rabbit antibodies to Notch2 (1:1000, ab118824, Abcam), TIM3 (1:500, ab185703, Abcam), mTORC1 (1:1000, ab25880, Abcam), CD9 (1:2000, ab92726, Abcam), CD63 (1:1000, ab134045, Abcam), Calnexin (1:20 000, ab92537, Abcam) and β‐actin (1:1000, ab8227, Abcam), and then re‐probed with IgG antibody complexed to horseradish peroxidase (1:2000, ab97051, Abcam). Enhanced chemiluminescence solution (ECL808‐25, Biomiga) was added on membrane for colour development. After scanning and with an optical luminometer (GE, Pittsburg, USA), the protein bands were analysed by Image Pro Plus 6.0 software (Media Cybernetics, Silver Spring, Maryland, USA) to analyse the relative expression levels of proteins with β‐actin as the internal reference.

Total proteins were extracted using the radioimmunoprecipitation assay lysis buffer (RIPA; R0010, solarbio) containing phenylmethanesulfonyl fluoride (PMSF), and the concentration was determined with a BCA protein assay kit (Thermo Fisher Scientific, NY, USA). 30 μg of total protein samples were subjected to polyacrylamide gel electrophoresis (PAGE) at 80 V for 35 min followed by 120 V for 45 min, then transferred into the polyvinylidene fluoride (PVDF) membranes (Amersham, USA). After being blocked in 5% skim milk for 1 h, the membranes were incubated with primary rabbit polyclonal antibodies overnight at 4 °C, then exposed to horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG H&L (ab6721, 1:3000, abcam, Cambridge, UK) at room temperature for 1 h. PBST buffer (PBS buffer supplemented with 0.1% Tween-20) was used to wash the membranes three times after each reaction. The primary antibodies included KPNA2 (ab84440, 1:1000, abcam, Cambridge, UK), E-cadherin (ab15148, 1:500, abcam, Cambridge, UK), N-cadherin (ab76057, 1:1000, abcam, Cambridge, UK), MMP-2 (ab37150, 1:1000, abcam, Cambridge, UK), MMP-9 (ab73734, 1:1000, abcam, Cambridge, UK) and GAPDH (ab9485, 1:2500, abcam, Cambridge, UK). Images were captured under an optical luminometer (GE, USA), and the protein bands were analyzed using the Image Pro Plus 6.0 software (Media Cybernetics, USA).

Total proteins were extracted from cells using RIPA lysis buffer (Invitrogen; Thermo Fisher Scientific, Inc.) and the concentration of proteins was measured by bicinchoninic acid (BCA) protein assay kit (Beyotime Institute of Biotechnology, Haimen, China). Protein samples were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF; Amersham, USA) membranes. After being blocked with 5% non-fat milk at room temperature for 1 h, the membranes were incubated with primary rabbit polyclonal antibodies Axin2 (ab32197, 1:1000, abcam, UK), β-catenin (ab32572, 1:5000, abcam, UK), c-Myc (ab39688, 1:1000, abcam, UK), cyclin D1 (ab16663, 1:200, abcam, UK) and GAPDH (ab9485, 1:2500, abcam, UK) overnight at 4 °C, followed by hybridization with horseradish peroxidase (HRP) -conjugated secondary antibody goat anti-rabbit IgG H&L (ab6721, abcam, UK) at room temperature for 1 h. The membranes were washed with PBST (PBS buffer containing 0.1% Tween-20) in triplicate with 10 min for each time before and after hybridization. Images of the protein bands were observed and captured under an optical luminometer (GE, USA).

Total protein was extracted, electrophoresed and then electroblotted to polyvinylidene fluoride membranes (Amersham, Piscataway, NJ), which were blocked with 5% skim milk powder for 1 h. After that, the membrane was incubated overnight at 4°C with the following primary antibodies to SP1 (ab227383, 1: 5000), cleaved PARP (ab32064, 1: 5000), cleaved Caspase-3 (ab49822, 1: 500), KDM2A (ab191387, 1: 1000), p-ERK (ab223500, 1: 400), ERK (ab17942, 1: 1000), and β-actin (ab179467, 1: 5000) as well as with horseradish peroxidase (HRP)-labeled secondary antibody goat anti-rabbit IgG (H&L, ab6721, 1: 3000) for 1 h at room temperature. After scanning and development with an optical luminometer (General Electric, Waukesha, WI), the gray value of the target band was quantified using the Image Pro Plus 6.0 software (Media Cybernetics). All the above-mentioned antibodies were purchased from Abcam (Cambridge, UK).

Total protein was extracted from cells. Protein concentration was measured using a bicinchoninic acid kit (Thermo). Protein (30 μg) was subjected to polyacrylamide gel electrophoresis at 80 V for 35 min and 120 V for 45 min. After the electrophoresis was completed, proteins were transferred to a polyvinylidene fluoride membrane. Membrane was blocked by 5% skim milk at ambient temperature for 1 h. Membranes were incubated with rabbit anti-FOXK1 (1:1,000, ab18196; Abcam), rabbit anti-Snail (1:1,000, ab180714; Abcam), rabbit anti-MMP-2 (1:1,000, ab97779; Abcam), rabbit anti-MMP-9 (1:1,000, ab73734; Abcam), rabbit anti-Bcl-2 (1:2,000, ab182858; Abcam), rabbit anti-Bax (1:2,000, ab32503; Abcam), rabbit anti-E-cadherin (1:500,000, ab76319; Abcam), rabbit anti-N-cadherin (1:2,000, ab18203; Abcam), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:2,500, ab9485; Abcam) at 4°C overnight. Membranes were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (1:10,000, ab6721; Abcam) for 1 h at ambient temperature after PBST washing. Membranes were washed three times with PBST buffer for 10 min each. After development with an optical luminometer (GE, Boston, MA, USA), gray intensity of protein bands was measured by Image ProPlus 6.0 (Media Cybernetics, Rockville, MD, USA).

The radioimmunoprecipitation assay (RIPA) buffer with 0.1% phenylmethylsulfonyl fluoride (PMSF) was used, and the cells were placed on ice for lysis for 20 min. After centrifugation (12,000 g, 4°C, and 10 min), the supernatant was transferred to a new EP tube to obtain the total protein products of cells. The protein concentration of the samples was determined by the BCA method, and protein was conducted according to different protein concentrations. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were immediately transferred to a polyvinylidene difluoride (PVDF) membrane treated with methanol. Under the condition of 4°C, the membrane was added with 5% skim milk powder and incubated overnight. Subsequently, the primary anti-ECT2 antibody (Abcam, UK) and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Abcam, UK) were added to the membrane and incubated at room temperature for 2 h. Then, the membrane was incubated for 30 min at the same condition of room temperature with the secondary antibody goat anti-rabbit IgG (Abcam, UK). GAPDH was used as the internal reference. An optical luminometer (GE, USA) was used for scanning and development; then, photos were taken.