Levels of total mTOR and phosphorylated protein at Ser2448 were measured in DMSO and calyculin A treated cells. Serum-starved HeLa cells were seeded on amine coated plates within the working range of the assay and treated with 10 nM calyculin A or DMSO control in serum free media for 15 minutes the day of fixation. Specificity of phosphorylation was determined by treating calyculin A-treated HeLa cells with LPP for 45 minutes at 40°C immediately after permeabilization. The relative levels of Total mTOR and mTOR pSer2448 are shown after background subtraction. Specific Phosphorylation was calculated by determining the ratio of pSer2448 to Total mTOR levels after normalization (bottom).
Levels of total mTOR and phosphorylated protein at Ser2448 were measured in serum-starved NIH3T3 cells that were treated with PDGF recombinant protein at 50 ng/mL in 1% BSA. The relative levels of Total mTOR and mTOR pSer2448 are shown after background subtraction. Specific phosphorylation was calculated by determining the ratio of pSer2448 to Total mTOR levels after normalization (bottom).
Levels of total mTOR and phosphorylated protein at Ser2448 were measured in calyculin A treated cells. Serum-starved HeLa cells were seeded on amine coated plates within the working range of the assay and treated with 10nM calyculin A in serum free media for 15 minutes the day of fixation. The relative levels of Total mTOR (Left) and mTOR pSer2448 (Right) are shown after background subtraction.
Calyculin A-treated HeLa Lysates were treated with ? Ppase (LPP) 34°C or left untreated on ice. Samples were then diluted in SDS-PAGE buffer and loaded at 40 µg/well. Membranes were blocked with 20% Blocking Buffer ab126587 in TBST for 1 hour and incubated with either the antibody mTOR pSer2448 or the antibody against total mTOR in 10% Blocking Buffer, in TBST overnight. Labeling was carried out with secondary antibodies conjugated to HRP. Treatment with calyculin A in HeLa cells induces phosphorylation at Ser2448 and ? Ppase completely dephosphorylates mTOR.
HeLa cells were seeded on glass coverslips and allowed to adhere overnight. After adhesion, cells were treated with DMSO or calyculin A for 15 minutes and fixed with 4% paraformaldehyde. The levels of mTOR total and mTOR pSer2448 were measured following the protocol as described above until step 11.5. The mouse mTOR total protein was labeled with goat anti-mouse-488 whereas the rabbit mTORpSer2448 was labeled with goat anti-rabbit-594. mTOR total and mTOR pSer2448 show specific co-localization in the cytoplasm. The cytoplasmic signal for the pSer2448 target is low on serum starved HeLa cells treated with DMSO only (A), whereas it is significantly induced with the use of 10 nM calyculin A (B). The pSer2448 specific signal is completely removed when calyculin A-treated HeLa cells are treated with LPP (C).