P pi3k

Prechilled RIPA buffer containing phosphatase inhibitors and protease inhibitors was used to lyse Cells and tissues. The protein lysates were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were blocked in 5% skimmed milk in 1x TBS-T (0.5% Tween-20) and incubated overnight at 4°C with the following primary antibodies: HRK (1:500; immunoway), CCND1, CDK4, CDK6, p21, BAX, Bcl-2, cleaved caspase-3, caspase-3, AKT, p-AKT(Ser473), p-PI3K(Tyr458), PARP1, cleaved-PARP1, GAPDH (1:1000; Proteintech), mTOR, p-mTOR(Ser2448), PI3K (1:1000; ZEN BIO). P70S6K, p-P70S6K(1-T389) (1:1000; Abclonal). After incubated with HRP-conjugated secondary antibodies for 1 h at room temperature, the membranes were visualized with ECL Western Blotting Substrate (FDbio). Immunoblotting signals were detected by densitometry using Image J software.

After drug treatment, cell/small intestine tissue was harvested and lysed with 1 × RIPA lysis buffer (Beyotime, Shanghai, China) containing phenyl-methyl-sulfonyl fluoride (PMSF, Beyotime, Shanghai, China). The proteins were obtained from cell lysates and separated on SDS-PAGE. After transferring proteins to PVDF membranes (Milipore, Darmstadt, Germany), the membranes were blocked with TBST supplemented with 5% skimmed milk (Biofroxx, Guangzhou, China) for 1 h at room temperature. The membranes were then incubated with the primary antibodies (TNFα, IL-1β, CRHR1, CRHR2, GAPDH, p-PI3K, PI3K, p-AKT, AKT, p-mTOR, mTOR, and ZO-1; 1:1000 dilution, Proteintech, Wuhan, Chian) at 4 °C overnight, followed by an incubation with HRP-conjugated secondary antibody (Proteintech, Wuhan, China) for 1 h at room temperature. Signals were detected using the ECL Western blotting substrate (Millipore, Darmstadt, Germany) and quantified with imageJ software (ImageJ 1.46r, National Institutes of Health, Bethesda, MD, USA). The data were obtained from three independent experiments.

HCASMC cells were distributed in six-well plates at 5 × 10⁵/dish, and the corresponding treatment was carried out for 24 h. Then, cell lysis was conducted with RIPA lysis kit (Beyotime) including phenylmethanesulfonyl fluoride. Quantification of total protein was detected with BCA protein assay kit (Beyotime). Total proteins (20 μg) were separated using 7.5–10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The resolved protein was subsequently transferred onto a nitrocellulose membrane. Membranes were blocked with quick blocking buffer (Beyotime) for 10 min at room temperature, and then incubated with primary antibodies at 4°C for 15 h, including p-STAT3, p-MAPK14, p-AKT (1:1,000; Cell Signaling Technology, Danvers, MA, USA), p-PI3K, PI3K, AKT, mTOR, and p-mTOR (1:1,000; Proteintech Group, Wuhan, China). Membranes were washed and then incubated in secondary antibodies (1:10,000) at room temperature for 1 h. Proteins were imaged by the Bio-Rad Chemi Doc XRS imaging system (Bio-Rad, USA).

The tissues that has been ground down or cells were treated with ristocetin-induced platelet aggregation buffer (Shandong Sparkjade Scientific Instruments Co., Ltd., Jinan, China) for total protein extraction. The BCA kit (Solarbio, Beijing, China) was then used for quantification. Subsequently, the protein samples (30–50 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF)membranes (Millipore). After blocking the PVDF membranes with 5% skim milk for two hours, they were rinsed twice using TBST (tris buffered saline + 0.5% [v/v] Tween-20) buffer. Subsequently, the membranes were incubated overnight at 4 °C after the addition of primary antibodies, including RRS1 (1:2000; Abcam; cat. No. ab188161), GRP78 (1:5000; Proteintech; cat. No. ab32072), GAPDH (1:2000; Abclonal; cat. No. AC002), phosphorylated (p)-AKT (1:1000; Abmart; cat. No. T40067), p-PI3K (1:1000; Proteintech; cat. No. AP0427), PI3K (1:1000; Abclonal; cat. No. A4992), AKT (1:1000; Abmart; cat. No. T55561), and ubiquitin (1:1000; Proteintech; cat. No. 10201-2-AP). After incubation with specific secondary antibodies (1:5000; Abclonal; cat. No. AS014 and AS003) for one hour at room temperature, the visualization of PVDF membranes were conducted based on electrochemiluminescence (ECL) (Abclonal, Wuhan, China).

Cultivate cells in a 24-well plate and fix with 4% paraformaldehyde for 15 min. Wash three times and permeabilize for 10 min in PBS containing 0.2% Triton X-100. Incubate overnight at 4 °C with mouse anti-human antibodies p-AKT and p-PI3K (Proteintech, China). The next day, wash with PBS and incubate for 2 h with goat anti-mouse IgG secondary antibody labeled with Alexa Fluor 488. After washing with PBS, incubate the cell samples with DAPI for 5 min and capture fluorescence images using a fluorescence microscope.