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. Author manuscript; available in PMC: 2016 Jan 31.
Published in final edited form as: Nat Immunol. 2015 Jun 29;16(8):838–849. doi: 10.1038/ni.3205

Figure 3. IFN-γ suppresses mTORC1 activation and downstream functions.

Figure 3

(a-c) Immunoblot analysis of whole cell lysates from control or IFN-γ-treated macrophages stimulated with Pam3CSK4 (10 ng/ml) for the indicated times and probed with antibodies against p-4E-BP1 (a, b) or p-p70S6K (c). In (b), mTOR inhibitors PP242 (50 nM ), Torin1 (50 nM) or Rapamycin (500 nM) were added for 30 min. (d) Immunoblot analysis of LC3A and LC3B in control or IFN-γ-primed macrophages. (e) Upper: Immunofluorescence images of LAMP1 (red) and mTOR (green) co-staining in control or IFN-γ-primed macrophages stimulated with Pam3CSK4 (10 ng/ml) for 4 h; nuclei were stained with DAPI (blue). Quantitation of co-localization (lower panel) between LAMP1 and mTOR; data are presented as mean ± SEM of the percentage of co-localized cells from 600 cells analyzed in three independent experiments; * p = 0.0001 by unpaired student t test. (f) Immunoblot analysis of HES1 in human primary macrophages pretreated for 30min with vehicle control DMSO or increasing concentrations of Rapamycin (0 nM, 500 nM, 1 μM), and then stimulated with Pam3CSK4 (10 ng/ml) for 0, 2, or 4h; p38α serves as loading control. Data are representative of at least three independent experiments (a-e).