Figure 2
From: Activity-independent targeting of mTOR to lysosomes in primary osteoclasts
Autophagy is inhibited in osteoclasts with R740S mutation. Spleen-derived osteoclasts were differentiated as described in “Materials and Methods”. (A) Immunoblotting, representative cropped blots of 4 independent experiments. 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. Whole cell lysates were separated on 4–20% gradient gels, transferred to a nitrocellulose membrane and probed for LC3, p62 and actin. (B) Quantification of immunoblots; combined normalized data of 4 independent experiments, mean ± std. dev; * indicates statistical significance compared to +/+ control, p < 0.05. (C) LC3 immunostaining. Spleen-derived osteoclasts were treated as described in (A), fixed, stained with anti-LC3B antibody as described in “Materials and Methods”; nuclei were stained with DAPI. Representative images. Bars, 17 μm.
