Anti pi3k

Western blotting was carried out as previously described with a minor modification. The antibodies used in the study are as follows: rabbit polyclonal anti-MMP2, anti-MMP7, anti-MMP9, anti-Bcl-xl, anti-Bcl-2, anti-Bax, anti-E-cadherin, anti-PI3K, anti-p53, anti-GSK3β, anti-Flot-1, anti-Akt3 (Proteintech, Wuhan, China), anti-phospho-Akt3 (S472) (Abgent, Suzhou, China), anti-phospho-p65 (S536), p50, anti-IκB, anti-Foxo1, anti-phospho-Foxo1 (S256), anti-p38, anti-p27, anti-p21, anti-CCNA1, anti-CCNE2 (Sangon Antibody R&D Center, Shanghai, China), anti-phospho-mTOR (S2448) (ImmunoWay, Newark, DE, USA), mouse monoclonal anti-Flot-2, anti-α-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-β-actin (Sigma, USA). Akt Inhibitor VIII, a specific Akt inhibitor, was purchased from Merck Millipore (Merck KGaA, Darmstadt, Germany). Quantification of signal intensity (IOD, integral optical density) was performed with Gel-Pro Analyzer software(Version 4.0). Expression change was indicated by IOD ratio of targeted protein before and after treatments. And the intensity was normalized by β-Actin signal. All detections were repeated for three independent times.

Same as the previous experiments, MOVAS were treated with starvation for 12 h. ET-1 inducted MOVAS of the pathological group for 24 h. And then, the corresponding drugs stimulated each group for 48 h. Protein was extracted from lysed cells. When we detected NF-κB, we extracted the nuclear protein from MOVAS. While assaying other factors, the total protein was extracted. The SDS-PAGE gel was prepared with a gel preparation kit (Servicebio, CHN). The samples of each group were separated by SDS gel electrophoresis and then transferred to nitrocellulose membranes, added the primary antibodies overnight after blocking and incubation, and incubated with secondary antibody for 1 h the next day. Protein signals were visualized by ChemiDoc System (BioRad). ImageJ 1.53 software was used to calculate the gray value. The main antibodies used in this study are anti-PI3K (Proteintech, CHN, 1 : 3000), anti-Akt (Proteintech, 1 : 1000), anti-phospho-Akt (Proteintech, 1 : 2000), anti-IκB-α (Cell Signaling Technology, USA, 1 : 1000), anti-NF-κB (Proteintech, 1 : 2000), anti-Bax (CST, 1 : 1000), anti-Bcl-2 (Proteintech, 1 : 1000), anti-GAPDH (Proteintech, 1 : 5000), anti-H3 (Proteintech, 1 : 1000), anti-iNOS (CST, 1 : 1000), anti-TNF-α (CST, 1 : 1000), anti-Rabbit IgG (Servicebio, 1 : 3000), and anti-Mouse IgG (Servicebio, 1 : 3000) [23 (link), 24 (link)].

The protein concentrations were determined using a BCA protein assay kit (Beyotime, Beijing, China). Proteins were electrophoresed on 10% SDS‐polyacrylamide electrophoresis gels and transferred onto a PVDF membrane (Millipore, Merck, Germany). The membranes were blocked in 5% skim milk for 4 h at room temperature. The following were used as primary antibodies: anti‐TLR4 (Santa Cruz Biotechnology, Dallas, TX, USA), anti‐p‐NF‐κB p65, anti‐p‐PI3K, anti‐STAT3, anti‐p‐STAT3 (Beyotime, Beijing, China), anti‐NF‐κB p65, anti‐p‐IκBα, anti‐IκBα, anti‐p‐mTOR, anti‐mTOR, anti‐p‐AKT, anti‐AKT (Cell Signaling Technology, Danvers, MA, USA), anti‐PI3K, anti‐cleaved Caspase 3, anti‐BAX, and anti‐BCL2 (Proteintech, Wuhan, China). Glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH; Proteintech, Wuhan, China) served as the loading control for total proteins. The blots were incubated with the primary antibodies overnight at 4 °C. After washing three times with TBST, the blots were incubated with anti‐mouse secondary antibodies and anti‐rabbit secondary antibodies (Beyotime, Beijing, China) for 1 h at room temperature. Western blots were performed for three independent experiments of each treatment. The intensity of each band was quantified with Image J software (Version 1.52p, NIH).

After digestion with trypsin (C0201, Beyotime, China), the cells were washed twice in phosphate-buffered saline (PBS), then lysed with protein-loaded buffer (20315ES05, YESEN, China) and boiled at 100 °C for 10 min. The separation of same amounts of proteins was in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes, and subjected to immunoblotting analysis with anti-GAPDH (60004-1-Ig, Proteintech, China), anti-METTL14 (26158-1-AP, Proteintech, China), anti-ETS1 (12118-1-AP, Proteintech, China), anti-YY1 (22156-1-AP, Proteintech, China), and anti-YTHDF1 (17479-1-AP, Proteintech, China), anti-YTHDF2 (24744-1-AP, Proteintech, China), anti-YTHDF3 (25537-1-AP, Proteintech, China), anti-Cyclin D1 (26939-1-AP, Proteintech, China), anti-Cyclin E1 (11554-1-AP, Proteintech, China), anti-PI3K (20584-1-AP, Proteintech, China), anti-phospho-PI3K (4228T, cell signaling, USA), anti-AKT (60203-2-Ig, Proteintech, China), anti-phospho-AKT(28731-1-AP, Proteintech, China). The polyvinylidene difluoride (PVDF) membrane was incubated with the secondary antibody at room temperature for one hour, followed by visualization using the ECL luminescent solution (P1050, Applygen, China) and the AI600 image gel imaging analyzer.

Different groups of GH3 and AtT20 cells were lysed on ice with RIPA buffer. The isolated exosomes can be directly mixed with the loading buffer. Then, protein lysates were electrophoresed on 10% SDS polyacrylamide gels, transferred onto PVDF membranes (Millipore, Burlington, MA, USA), and blocked with NcmBlot blocking buffer (NCM Biotech, Suzhou, China). Afterward, primary antibodies: anti-ONECUT2 (Biorbyt, Cambridge, UK, cat: orb49419, 1:1000), anti-PI3K (Proteintech, Wuhan, China, cat: 60225-1-Ig, 1:1000), anti-P-PI3K (Thermofisher, Waltham, MA, USA, cat: PA5-104853, 1:1000), anti-PKCδ (Proteintech, Wuhan, China, cat: 14188-1-AP, 1:1000), anti-P-PKCδ (Proteintech, Wuhan, China, cat: 29562-1-AP, 1:1000), anti-mTOR (Protentech, Wuhan, China, cat: 66888-1-Ig, 1:1000), anti-P-mTOR (Proteintech, cat:67778-1-Ig, 1:1000), anti-AKT (Proteintech, cat: 60203-2-Ig, 1:1000), anti-P-AKT (Proteintech, Wuhan, China, Cat: 80455-1-RR, 1:1000), anti-ERK (Proteintech, Wuhan, China, cat: 11257-1-AP, 1:1000), anti-P-ERK (Proteintech, Wuhan, China, cat: 80031-1-RR, 1:1000), anti-GAPDH (Proteintech, Wuhan, China, Cat: 60004-1-Ig, 1:1000), anti-CD63 (Santa Cruz, CA, USA, cat: sc-5275;1:200), anti-CD9 (Santa Cruz, CA, USA, cat: sc-13118; 1:200) were added at 4 °C overnight. Subsequently, secondary antibodies (1:5000; Proteintech, Wuhan, China) were incubated for 2 h.