Figure 4.

TFEB is regulated post-translationally by RANKL. (A) qPCR expression analysis of the indicated genes in bone marrow-derived osteoclasts differentiated in the presence of M-CSF and RANKL for 0–6 d. The expression level of each gene is normalized to the expression at day 6. (B) RAW 264.7 cells stably transfected with TFEB-Flag were treated for the indicated times with RANKL (50 ng/mL). Nucleus and cytosol extracts were prepared as described in the Materials and Methods, and TFEB protein was revealed by Western blotting using Flag antibodies. Lamin C and tubulin were used as nuclear- and cytosolic-specific markers, respectively. (C) Bone marrow-derived monocytes were treated with RANKL (50 ng/mL) for the indicated times, and endogeous TFEB protein was detected by Western blotting. (D) RAW 264.7 cells stably transfected with TFEB-Flag were treated with cycloheximide (CHX at 100 μg/mL) and/or RANKL (50 ng/mL) for the indicated times, and TFEB protein in total cell extracts was revealed by Western blotting using Flag antibodies. (E) RAW 264.7 cells stably transfected with TFEB-Flag were treated with RANKL (50 ng/mL) for the indicated times, and TFEB protein in total cell extracts was revealed by Western blotting using Flag antibodies. (F) RAW 264.7 cells stably transfected with TFEB-Flag were treated with RANKL (50 ng/mL) for 30 min. The total cell extracts were next subjected to λPPase treatment as described in the Materials and Methods. In D–G, actin was used as a loading control. In all experiments, the cells were cultured in the presence of FBS (10%).