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. Author manuscript; available in PMC: 2010 Jun 1.

Published in final edited form as: Free Radic Biol Med. 2009 Mar 10;46(11):1500–1509. doi: 10.1016/j.freeradbiomed.2009.03.001

Fig. 5 — Modulation of mTORC1 and mTORC2 assembly by H₂O₂. Serum and leucine starved cells were lysed in CHAPS non-reducing buffer following treatment with 250 μM H₂O₂ and stimulation with leucine (0.8 mM), insulin (174 nM), or both for 30 min. Equal amounts of protein were then incubated with an anti-mTOR antibody and immune complexes separated using magnetic beads. Intact complexes were resolved via SDS-PAGE, transferred to PVDF membranes, and immunoblotted for mTOR, rictor, raptor, and SIN1. Immunoblots are representative of 6 individual experiments and histograms depict relative densities of each complex component relative to its binding partner. Data are presented as means ±SE. “*” identifies statistically significant differences between insulin or leucine+insulin and vehicle control, while “” signifies an effect of H₂O₂.

Inline graphic — Modulation of mTORC1 and mTORC2 assembly by H₂O₂. Serum and leucine starved cells were lysed in CHAPS non-reducing buffer following treatment with 250 μM H₂O₂ and stimulation with leucine (0.8 mM), insulin (174 nM), or both for 30 min. Equal amounts of protein were then incubated with an anti-mTOR antibody and immune complexes separated using magnetic beads. Intact complexes were resolved via SDS-PAGE, transferred to PVDF membranes, and immunoblotted for mTOR, rictor, raptor, and SIN1. Immunoblots are representative of 6 individual experiments and histograms depict relative densities of each complex component relative to its binding partner. Data are presented as means ±SE. “*” identifies statistically significant differences between insulin or leucine+insulin and vehicle control, while “” signifies an effect of H₂O₂.