Anti akt3

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.

Protease inhibition was used to extract total protein from cell lysis of MKN-45 and MGC-803. Bicinchoninic acid protein assay kit was used to measure protein concentration. Protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis in appropriate concentration and transformed onto polyvinylidene fluoride membranes. After blocking for 1 h at 4 ℃ using tris-buffered saline with Tween^® 20 (TBST) brewed skim milk powder, the membrane was incubated overnight with the anti-NEK7 (ab13514, abcam, UK) antibody and anti-GAPDH (ab8245, abcam) antibody, which was diluted to an appropriate concentration. Then, after washing, the membrane was incubated with the second antibody at 4 ℃ for at least 1 h and washed by TBST three times. The anti-CDK4 (Cat No. 11026-1-AP), anti-CCND2 (Cat No. 10934-1-AP), anti-KIF3A (Cat No. 13930-1-AP), anti-AKT3 (Cat No. 21641-1-AP), and anti-PRKG1 (Cat No. 21646-1-AP) antibodies were purchased from the Proteintech Group (IL, USA). Signals were detected using a chemiluminescence system (SensiCapture imaging system, Peiqing Technology Co. LTD, China).

Sections from adrenocortical adenomas and adjacent tissue arrays were deparaffinized, rehydrated in various alcohol grades, and then microwave oven treated (15 min in 0.01 M-citrate buffer at pH 6.0). The tissue sections were reacted with anti-TBX3 (Santa Cruz, Sc-166623, dilution 1:50; USA), anti-SGK1 (Santa Cruz, sc-28338, dilution 1:50; USA), anti-AKT3 (Proteintech, 21641-1-AP, dilution 1:50; China) or anti-SOX2 (Proteintech, 11064-1-AP, dilution 1:50; Sigma–Aldrich) antibodies overnight at 4 °C. Immunohistochemical staining was done as previously described [7 (link)]. Immunofluorescence staining was performed for Ki-67 (diluted 1:100; GB111141; Servicebio, Wuhan, China), DAPI (diluted 1:5000, C1002, Beyotime). Immunofluorescence staining was done as previously described [27 (link)].