Akt kinase activity assay kit

The nominated protein kinase activity inhibition by the novel compounds was determined quantitively by the VEGFR2 (KDR) Kinase Assay Kit (Catalog # 40325, BPS, Bioscience, San Diego, CA, USA) against VEGFR-2 [55 ,56 (link)] and Akt Kinase Activity Assay Kit (ab139436, ABCAM, USA) for AKT-1 enzyme on cell lysate according to the reported procedures [57 ,58 (link)]. The outcomes were expressed as IC₅₀ values (mean standard deviation) calculated from the dose–response curves and their linear regression equations. Sorafenib was used as the reference in the VEGFR-2 assay, and LY2780301 was used in the AKT assay.

The IL-8 Human ELISA Kit (KHC0081; Thermo Fisher Scientific, Waltham, MA, USA) was employed to determine the amount of interleukin-8 in the culture medium; 50 µL of the culture medium was used. Absorbance at 450 nm was recorded by microplate reader Infinite F200 (Tecan, Seestrasse, Switzerland). AKT Kinase Activity Assay Kit (ab139436; Abcam, Cambridge, UK) was used for determining AKT kinase activity. The signal was developed according to the manufacturer’s instructions. Absorbance at 450 nm was recorded by microplate reader Infinite F200 (Tecan, Seestrasse, Switzerland).

The in vitro inhibition of PI3K and Akt kinase activities by VIIb was assessed using a PI3Kα (p110α/p85) assay kit (Catalog #79781; BPS Bioscience, Inc, San Diego, CA, USA), PI3Kγ (p110γ/PIK3R5) assay kit (Catalog #79803; BPS Bioscience, Inc, San Diego, CA, USA), and Akt Kinase Activity assay kit (ab139436; Abcam, Cambridge, UK) following the manufacturer’s instructions as described previously [58 (link),59 (link)]. The results were expressed as IC₅₀ values using dose–response curves and linear regression equations. LY294002 compound was taken as the reference compound. The values were means ± sd; n = 3.

The Akt Kinase Activity Assay kit (ab139436, Abcam) was used to detect total Akt activity in gastrodin-treated OSCC cells according to the manufacturer’s protocols. In brief, lysates from gastrodin-treated CAL27-CR and SCC4-CR cells were homogenized, followed by incubation with ATP and Akt phosphor-specific substrate antibodies in an ELISA plate. Subsequently, each well was supplemented with HRP-conjugated anti-rabbit IgG and TMB solution, followed by treatment with a stop solution. The absorbance was finally measured at a wavelength of 450 nm.

The AKT kinase activity assay kit (Abcam) was used to quantitate the activity of AKT according to the manufacturer’s instructions as we reported recently [44 (link)]. In brief, the assay is based on a solid-phase ELISA. A specific synthetic peptide is used as a substrate for AKT along with a polyclonal antibody that binds the phosphorylated substrate.

Serum insulin assays were performed using a standard ELISA kit (cat# EZRMI-13K, Millipore, USA) per the manufacturer’s instructions. Tissue Akt activity was measured from tissue lysates using the Akt Kinase Activity Assay Kit (ab139436, Abcam, USA) as manually instructed. Blood glucose, triglyceride, and cholesterol were examined by Abbott Auto-analyzer (Ci8200).

The AKT kinase activity assay kit (Abcam) was used to quantitate the activity of AKT according to the manufacturer’s instructions as we previously reported [40 (link),76 (link)]. In brief, the assay is based on a solid phase ELISA. A specific synthetic peptide is used as a substrate for AKT along with a polyclonal antibody that binds the phosphorylated substrate.