Anti akt1

The cells were washed three times with PBS and fixed with 4% paraformaldehyde for 30 min. After and additional three washes with PBS, the cells were incubated in 0.2% Triton-X-100 for 10 min. 5% goat serum used for blocked for 1 h, which was followed by three washes with PBS. Next, the cells were treated with anti-PAK1 (Abcam, 1:100) and anti-AKT1 (Proteintech, 1:50) for 4 °C and incubated overnight. Then the cells were incubated with a fluorescein binding secondary antibody (CST, 1:200) at room temperature for 1 h. The cell nucleus was stained for 30 s with 4,6-diamino-2-phenylindole (DAPI, Solarbio). A confocal fluorescence microscope (60 ×) was used to acquire images.

Medium 199 (M199), Hank's Balanced Salt Solution (HBSS), fetal bovine serum (FBS), and endothelial cell growth supplement were purchased from Invitrogen (Burlington, ON). VEGF-A was from Peprotech (Princeton, NJ). Rapamycin, PP242, a highly specific mTOR active-site inhibitor [12 (link)], and anti-tubulin-α was from Millipore (Temecula, CA). Ku-0063794, a second specific mTOR inhibitor [13 (link)] was from Sigma (St. Louis, MO). Anti-Akt1 was from Protein Tech (Chicago, IL). Hiperfect, non-silencing short interfering RNA (siRNA) and Akt1 silencing siRNA were from Qiagen Inc (Mississauga, ON). Human tumor necrosis factor-α (TNFα) was from Cedarlane (Mississauga, ON). Cycloheximide, phalloidin-FITC, anti-vinculin, and DAPI were from Sigma. Anti-S6K was from Abcam (Cambridge, UK). Anti-phospho-Akt^S473, anti-phospho-S6K^T389, anti-FAK, anti-phospho-FAK^Y397, anti-raptor, anti-rictor, and rictor siRNA were from Cell Signaling Technology (Danvers, MA). ON-TARGETplus human raptor siRNA-SMARTpool was from Thermo Scientific (Waltham, MA).

Antibodies used in this article include anti-TRAF4 (Proteintech: Wuhan, China, CAS: 66755-1-Ig), anti-TRAF4 (Abcam: NY, USA, CAS: ab245666), anti-Tubulin (Proteintech: Wuhan, China, CAS: 66031-1-Ig), anti-E-cadherin (Proteintech: Wuhan, China, CAS: 20874-1-AP), anti-MMP2 (Proteintech: Wuhan, China, CAS: 10373-2-AP), anti-β-catenin (Cell Signaling Technology: Danvers, MA, USA, CAS: 8480), anti-CCND1 (Proteintech: Wuhan, China, CAS: 60186-1-Ig), anti-GSK3β (Proteintech: Wuhan, China, CAS: 22104-1-AP), anti-p-GSK3β (Cell Signaling Technology: Danvers, MA, USA, CAS: 5558), anti-AKT1 (Proteintech: Wuhan, China, CAS: 60203-2-Ig), anti-p-AKT (Cell Signaling Technology: Danvers, MA, USA, CAS: 13038), anti-SETDB1(Cell Signaling Technology: Danvers, MA, USA, CAS: 2196), anti-Flag (Proteintech: Wuhan, China, CAS: 66008-3-Ig), anti-Flag (Proteintech: Wuhan, China, CAS: 80010-1-RR), anti-MYC-tag (Proteintech: Wuhan, China, CAS: 60003-2-Ig), anti-MYC-tag (Proteintech: Wuhan, China, CAS: 16286-1-AP), and anti-HA (Proteintech: Wuhan, China, CAS: 51064-2-AP).
Reagents used in this article including Cycloheximide (CHX) (Merck: Kenilworth, NJ, USA, CAS: 66-81-9) and proteasome inhibitor (MG-132) (MCE: Chongqing, China, CAS: 133407-82-6).