Phosphorylated akt thr308

Cells were cultured in 6‐well plates in complete medium and pretreated with GNF‐2 in serum‐free medium for 3 hours before induction with either M‐CSF (100 ng/mL) or RANKL (200 ng/mL) for the indicated times. BMMs and RAW 264.7 cells were treated with 2 µM and 5 µM GNF‐2, respectively. Western blot was performed as described previously.38 The primary antibodies used in this study were as follows: rabbit polyclonal phosphorylated Akt (Thr308) (1:1000, Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal Akt (pan) (1:1000, CST), rabbit polyclonal Erk1 (Thr202/Tyr204) + Erk2 (Thr186/Tyr187) (1:100, Abcam, Cambridge, MA, USA), rabbit polyclonal Erk1/2 (1:1000, CST), and rabbit monoclonal Gapdh (1:2000, CST). Densitomety analysis was performed using ImageJ39 and normalized to the Gapdh intensity. Relative phosphorylation of Akt and Erk was presented as the ratio between the phosphorylated normalized to the nonphosphorylated/total protein.

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).