M csf elisa

All cell incubations were performed in culture medium consisting of αMEM with 2 mM l-glutamine, 10% heat-inactivated FBS, 100 IU/ml Penicillin and 100 IU/ml Streptomycin (Life Technologies, 10007D). Mouse soluble RANKL (R&D Systems, 462 TR) and M-CSF ELISA (R&D Systems, DY416), RANKL ELISA (R&D Systems, DY462), and TRAF6 inhibitor (Novus, NBP2-26506) were used for in vitro experiments. CMG14-12 (CMG) media was generated as described before [24 ]. Collagenase type II (Calbiochem, 234155), dispase (Stem Cells Technologies, 07923), ascorbic acid (Sigma, A8960) and dexamethasone (Sigma, D4902) were used for osteoblast differentiation assays. AlamarBlue solution (Life Technologies, DAL1025) was used for cell viability assay. For western blot analysis and immunofluorescence staining, anti-IκBα (Cell Signaling, 4812), anti-phospho-IκBα (Cell Signaling, 2859P), anti-NF-κB p65 (Cell Signaling, 4764), and anti-phospho-NF-κB p65 (Cell Signaling, 3033P), anti-Src (Millipore, 05–184), and phospho-Src (Millipore, 07–909), anti-WDFY3 (Novus, NBP1-03332), anti-TRAF6 (Santa Cruz Biotechnology, clone D-10), anti-β-actin antibody (Cell Signaling, 4970), 800 or 680 secondary antibodies (Li-Cor). For flow cytometry, APC anti-mouse c-Fms and PE anti-RANK antibodies (Biolegend) were used for cell staining.

All cell incubations were performed in culture medium consisting of αMEM with 2mM L-glutamine, 10% heat-inactivated FBS, 100 IU/ml Penicillin and 100 IU/ml Streptomycin (Life Technologies, 10007D). Mouse soluble RANKL (R&D Systems, 462TR) and M-CSF ELISA (R&D Systems, DY416), were used for in vitro experiments. CMG14-12 (CMG) media was generated as described before [22 ]. Anti-Wdfy3 (Abnova, clone 2F12), anti-WDFY3 (Novus, NBP1-03332), anti-SQSTM1 (Progen, GP62-C), LC3 antibody (Novus, NB100-2220), anti-β-actin antibody (Cell Signaling, 4970), 800 or 680 secondary antibodies (Li-Cor) were used for in vitro experiments. Cyto-ID kit (Enzo biochem, ENZ-51031) was used for autophagosome staining. LysoTracker DND-99 was used for lysosome staining (Life Technologies, L-7528).