Ab8805

Proteins from vascular tissue and cultured VSMCs were lysed by protein lysis buffer. Proteins were run on 8–10% SDS-PAGE to separation and then electro-transferred to PVDF membranes (Millipore). The membranes were blocked with 5% milk in TTBS for 2 h at room temperature and incubated overnight at 4 °C using the primary following antibodies: anti-IL-1β (1:1000, Proteintech, 16,806–1-AP), anti-IL-6 (1:1000, Proteintech, 21,865–1-AP), anti-TNF-α (1:1000, Proteintech, 6029–1-Ig), anti-PTEN (1:1000, Abcam, ab32199), anti-pan-AKT (1:1000, Abcam, ab8805), anti-pan-AKT (phospho T308; 1:1000, Abcam, ab38449), and 1:1000 anti-β-actin (1:2000, Santa, sc-47778). In the next day, after washing in the TTBS for three times, membranes were incubated with a 1:5000 dilution of anti-rabbit or anti-mouse antibody (Santa Cruz) for 1 h at room temperature. Protein bands were detected by enhanced chemiluminescence (ECL) Fuazon Fx (Vilber Lourmat).

Total proteins from cells and tissues were extracted by the RIPA lysate. Protein quantification was conducted with a BCA protein quantification kit. An appropriate amount of protein sample was denatured at 100°C for 3–5 min and then subjected to protein electrophoresis using 10% SDS-PAGE for 1 h. the samples were transferred to PVDF membranes. The membranes were blocked in Tris Buffered Saline with Tween 20 blocking solution containing 5% skim milk powder at room temperature for 2 h. The membranes were washed three times with TBST and incubated overnight at 4°C using primary antibodies against MMP-2, MMP-9, p-PI3K, PI3K, p-AKT, AKT, and GAPDH (ab92536, ab76003, ab278545, ab140307, ab192623, ab8805, ab8245; all from Abcam, Shanghai, China). The next day, the horseradish peroxidase-labeled goat anti-rabbit secondary antibody IgG (ab96899) and goat anti-mouse secondary antibody IgG (ab96879) dilution buffer were added, and membranes were incubated with these secondary antibodies at room temperature for 2 h. The enhanced chemiluminescence reagent was added dropwise, and the membranes were imaged by the gel imaging system. The gray value of each band was analyzed by the ImageJ software. The results were expressed as the ratio of the gray value of the target protein to GAPDH, the internal reference protein.

Total proteins were extracted from CRC tissue and cell samples by the RIPA reagent obtained from Sigma, USA, and separated using 10% SDS-PAGE. The isolated proteins were transferred into PVDF membranes followed by 5% nonfat milk incubation to minimize the noise. Next, the membranes were immersed in milk containing primary antibodies against SOCS3 (Rabbit, 1:2000, ab16030, Abcam, UK), CD133 (Rabbit, 1:1000, ab216323, Abcam), SOX2 (Mouse, 1:2000, ab171380, Abcam), OCT4 (Rabbit, 1:5000, ab109183, Abcam), AKT (Rabbit, 1:500, ab8805, Abcam), STAT3 (Mouse, 1:5000, ab119352, Abcam), p-AKT (Rabbit, 1:1000, ab38449, Abcam), p-STAT3 (Rabbit, 1:5000, ab76315, Abcam), and GAPDH (Rabbit, 1:3000, ab124905, Abcam), and incubated overnight. After excess primary antibodies were washed off by PBS, the membranes were probed by HRP-conjunct secondary antibodies, and the signals were visualized using the ECL reagent (Bio-Rad).

Total proteins was extracted from tissues and then determined for protein concentration with a BCA kit (Boster Biological Technology Co.Ltd). Next, loading buffer was added into the proteins for 10 min of heating at 95 °C, followed by the sample loading of 50 μg per hole. Electrophoresis with 10% polyacrylamide gel (Boster Biological Technology co.ltd) was used to separate proteins, which were transferred to the PVDF membrane and blocked for 1 h with 5% BSA at room temperature. Next, primary antibodies (1:500 diluted) were added for overnight reaction at 4 °C, including RICTOR (ab70374, Abcam), p-Akt (s473) (ab8932, Abcam), Akt (ab8805, Abcam), and GAPDH (ab181602, Abcam). The membrane with proteins on it was washed for three times, 5 min each time, before the addition of secondary antibodies (1:1000 diluted, Abcam) for 1 h of incubation at room temperature. Subsequently, the membrane was washed again for three times/5 min before the development with ECL chemiluminescence reagent. With GAPDH as the internal reference gene, the gray value of target bands was analysed with the software Image J.

Rat endometrium tissues were initially homogenized and lysed in radio-immunoprecipitation assay (RIPA) buffer (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) supplemented with proteinase inhibitor (Lot#4693116001, Roche). Lysates were then centrifuged for 20 min at 12,000 g. The bicinchoninic acid (BCA) (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China) was used for quantification of protein concentration. Equivalent amounts of protein (20 μg) were then used for western blot with primary antibodies of VEGF (ab53465, Abcam, UK), PI3K (ab191606, Abcam, UK), P-PI3K (ab182651, Abcam, UK), AKT (ab8805, Abcam, UK), P-AKT (ab38449, Abcam, UK), Ang-1 (ab102015, Abcam, UK), Ang-2 (ab155106, Abcam, UK) and HIF-1a (ab1, Abcam, UK) at 4 °C with gentle shaking overnight. After washing with TBS-T for three times, the membranes were then incubated with the secondary antibody at RT for 1 h and detected using an ECL plus kit (Beijing Dingguo Changsheng Biotechnology Co. Ltd., China). The ECL signals were detected with Quantity One software (Bio-Rad, Hercules, CA) and blot intensities were quantified by using imageJ (NIH, Bethesda, MD) software. Tubulin (ab8245, Abcam, UK) was used as an internal control to validate the amount of protein loaded onto the gels.

After the behavioral experiment, three mice were randomly selected from each group for Western blotting. The brain tissue of the mice was quickly removed and the hippocampus tissue was separated at low temperature. The treated brain tissue requires extraction of histones at low temperature, followed by grinding and centrifugation of the sample. The protein concentration of the sample is determined from the standard curve and then heated to denature the proteins of the sample. Electrophoresis and membrane transfer are then performed. We use the first antibody to react with the PVDF membrane overnight, put down the second antibody the next day and finally rinse with PBS solution. The gels were then scanned using an automated gel imaging system and analysed for calculation using ImageJ software. Main antibodies and dilution ratio: Anti-Akt (ab81283, Abcam, 1:1000); Anti-beta Amyoid 1-42 (ab201060, Abcam, 1:1000); Anti-IL-1beta (ab9722, Abcam, 1:800); Anti-IL-10 (ab9969, Abcam, 1:800); Anti-PI3K (ab191606, Abcam, 1:1000); Anti-GFAP (BA0056, BOSTER, 1:800); Anti-IBA-1 (PB0517, BOSTER, 1:800); Anti-Beclin1 (ab207612, Abcam, 1:1000); Anti-LC3 (ab192890, Abcam, 1:1000); Anti-p-Akt (ab8805, Abcam, 1:1000); Anti-β-actin (GB12001, Servicebio, 1:2000).