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. 2013 Apr 15;27(8):955–969. doi: 10.1101/gad.213827.113

Figure 5.

Figure 5.

RANKL-induced TFEB stabilization is PKCβ-dependent. (A) RAW 264.7 cells stably transfected with TFEB-Flag were treated with vehicle (DMSO) or the indicated inhibitors as described in the Materials and Methods and with or without RANKL (50 ng/mL) for 16 h. TFEB protein in total cell extracts was determined by Western blotting using Flag antibody. (B) Relative qPCR expression analysis of PKCs encoding genes in primary monocytes and osteoclasts. Values are expressed as fold of Prcka expression levels in monocytes. (C,D) RAW 264.7 cells stably transfected with TFEB-Flag were treated with vehicle (DMSO) or the indicated inhibitors and with or without RANKL (50 ng/mL) for 16 h. TFEB accumulation in cell extracts was measured by Western blotting using Flag antibody. In all experiments, the cells were cultured in the presence of FBS (10%). (E) Bone marrow-derived monocytes were serum-starved for 3 h and treated for the indicated times with RANKL (50 ng/mL), and PKCβ phosphorylation was assessed by Western blotting using an antibody previously validated using Prkcb−/− cell extracts (see Supplemental Fig. S5C).