Figure 1
From: Activity-independent targeting of mTOR to lysosomes in primary osteoclasts
mTORC1 activity is increased in R740S/R740S cells. Spleen-derived osteoclasts were differentiated as described in “Materials and Methods”. On day 4 of culture, the cells were incubated with HBSS (starvation) for 60 min, and then in fully supplemented media for additional 30 min. (A) Immunofluorescence. Cells were fixed and stained with an anti-a3 antibody as described in “Materials and Methods”; nuclei were stained with DAPI. Representative images of three independent experiments. Bars, 15 μm. (B) Immunoblotting, representative cropped blots of 4 independent experiments. Whole cell lysates were separated on 4–20% gradient gels, transferred to a nitrocellulose membrane and probed for mTOR, p-mTOR, p-AKT, pan-AKT, p-p70S6K, and actin. (C) Quantification of immunoblots; combined normalized data of at least 4 independent experiments, mean ± std. dev; * indicates statistical significance compared to +/+ control, # indicates statistical significance compared to +/R740S control, p < 0.05.
