Akt kinase assay kit

Total RNA was extracted from mouse skeletal muscle with the use of an RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). Quantitative reverse transcription (RT) and PCR analysis was performed as described previously^{8 (link)}, with 36B4 mRNA as an internal control and with the use of an ABI StepOne Plus Real-Time PCR system (Applied Biosystems, Waltham, MA). The sequences of primer pairs are available on request. For immunoblot analysis, the protein concentration of cell or tissue extracts was measured with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA), and the same amount of protein was loaded in each lane. Primary antibodies included those to the following proteins: PDK1 (#3062), Akt (#9272), Thr³⁰⁸-phosphorylated Akt (#9275), Ser⁴⁷³-phosphorylated Akt (#9271), S6K (#9202), Thr³⁸⁹-phosphorylated S6K (#9205), S6 (#2217), Ser²³⁵- and Ser²³⁶- phosphorylated S6 (#2211), all from Cell Signaling Technology (Danvers, MA), as well as Thr²²⁹-phosphorylated S6K (1015B) from R&D Systems (Minneapolis, MN). Akt activity was measured with an Akt Kinase Assay Kit (#9840, Cell Signaling Technology).

Akt kinase assays were carried out using a non-radioactive Akt kinase assay kit (9840; Cell Signaling Technology, Danvers, MA) according to the manufacturer’s instructions. In vitro transcription/translation (IVT; L5010, Madison, WI) Flag-TSC2 protein was synthesized and used as the substrate.

A549 and H1299 cells were seeded into 10 cm culture dish and infected with lentivirus containing PRMT5 shRNA or scramble shRNA. After 48 hours, the cells were selected with puromycin and the stable depletion of PRMT5 cells were subjected to Akt kinase activity assay by Western blot analysis. The Akt kinase activity was detected by an Akt kinase assay kit (non‐radioactive) according to the manufacturer’s instructions (cat no. 9840; Cell Signaling Technology).

The AKT Kinase Assay kit was purchased from the Cell Signaling (Danvers, MA, # 9840). As instructed by the provided protocol, immobilized AKT antibody was used to immune-precipitate AKT from the indicated cell extracts [48 (link)]. Immune-precipitated pellets were then incubated in the Kinase Buffer containing GSK-3 fusion protein and cold ATP. Levels of GSK-3 phosphorylation using the anti-phospho-GSK-3α/β (Ser21/9) antibody were measured by western blotting and chemiluminescent detection. All the experimental steps were carried out strictly according to the instructions of the kit. Each experiment was repeated for three times.

AKT kinase assays were carried out using the non-radioactive AKT Kinase Assay Kit from Cell Signaling Technologies (Catalog #9840) according to the manufacturer's instructions with the following modification: HA-Tag antibody Sepharose bead conjugate was used to isolate AKT kinase. Briefly, HCT116 AKT1/2 DKO cells expressing exogenous HA-tagged AKT alleles were lysed in 1× lysis buffer and sonicated. Sonicated lysates were normalized to equal amounts of total protein. AKT was then purified from normalized lysates by HA immunoprecipitation for 3 hr at 4°C. HA beads were then washed two times with 500 µl lysis buffer and once with 1× kinase buffer. Beads were then resuspended in 50 µl kinase buffer, and kinase reaction was carried out for 30 min after addition of 1 µg of substrate and ATP to a final concentration of 200 µM. Reaction was terminated by adding 3× Laemmli buffer. 10 µl of kinase reaction were analyzed by immunoblot. Substrate phosphorylation and AKT content in each reaction was measured by immunoblot.