Phospho mtor ser2448 antibody

To determine the changes in the phosphorylation status of serine-²⁴⁴⁸ of mTOR, cardiac tissue lysates were subjected to Western blotting as described previously^{39 (link),44 (link)}. mTOR and phospho-mTOR (Ser²⁴⁴⁸) antibody were purchased from Cell Signaling Technology, highlighted. Tris-buffered saline-Tween 20 (TBST) containing 5% bovine serum albumin (BSA) was used for blocking the Western blots (PVDF) for one hour. Primary antibodies were diluted 1:1000 in 5% BSA in TBST. Blots were incubated for overnight at 4 °C in primary antibodies, washed with TBST, and were incubated in the horseradish peroxidase-conjugated secondary antibody (1:25,000 dilution in 5% BSA in TBST). After TBST washes, chemiluminescent substrate (Supersignal West Femto Maximum Sensitivity Substrate kit; Thermo Scientific) was added to visualize antibody binding using Bio-Rad ChemiDoc XRS image-analysis system. Quantitation of pSer²⁴⁴⁸-mTOR band density compared to total mTOR protein band density was performed and ratios were calculated. All protein band density quantifications were performed using Quantity One software (Bio-Rad Laboratories Inc. Berkeley, Ca). Data are reported as the normalized protein band density in arbitrary units.

Western blots were performed as previously described [37 (link)]. The antibodies used for these analyses were the following: GPRC6A antibody (ASSAY BIOTECHNOLOGY, #G321); Phospho-p44/42MAPK (ERK1/2)(Thr202/Tyr204) antibody (Cell Signaling,#9101); p44/42MAPK (ERK1/2) (Cell Signaling,#9102); Phospho-mTOR (Ser2448) antibody (Cell Signaling,#2971); Phospho-Akt (Ser473) antibody (#9271).