Fig. 1
From: Pasteurella multocida toxin- induced osteoclastogenesis requires mTOR activation
The mTOR pathway is activated in response to PMT. RAW264.7 cells were stimulated with PMT (5 nM) w or w/o rapamycin (10 ng/ml) for the indicated period of time or were left unstimulated, treated with the solvent control DMSO or rapamycin only. Cells were lysed and the proteins immunoblotted. (a) mTOR Ser2448 phosphorylation was determined using a specific antibody and β-actin was used as loading control. (b) To investigate the uptake and intracellular action of PMT, cells were stimulated for the indicated time-points with 5 nM PMT and lysed. As a control, cells were stimulated with a catalytically inactive mutant of PMT (PMTC1165S). The immunoblot was probed with a specific antibody detecting the deamidated form of Gαq (Q209E) or total Gαq. (c, d) mTOR-dependent protein phosphorylation from RAW264.7 cells stimulated with PMT was detected using specific antibodies for PRAS40 (Thr246) or p70 S6K1 (Thr389); ß-actin was used as a loading control. The results shown are one representative example of three independent experiments