Lamp2

Immunoblot analyses were performed using the following antibodies: Akt, Aktp^S473, p70S6K, phospho-p70S6K, 4EBP1, ERK1/2, Atg5, ULK, ERK1/2, phopho-ERK1/2, p38, phospho-p38, phospho-NFκB and phospho-4EBP1 were all purchased from Cell Signaling. FAK (Santa Cruz Biotechnology, Inc.), actin (Cytoskeleton), LC3 (Novus) and FAKp^Y397 (BD Transduction Laboratories) were purchased from the suppliers indicated. Monoclonal anti-HA (16B12) and polyclonal anti-HA (HA.11) were both purchased from Covance. For immunofluorescence studies, LC3 (Millipore), mTOR (Cell Signaling), LAMP-1 and LAMP-2 (Developmental Studies Hybridoma Bank, University of Iowa), p62 (abcam) and ubiquitin (FK2, Enzo Life Sciences) were from the suppliers indicated. Secondary antibodies included goat anti-mouse-Cy3, donkey anti-rabbit Alexa Fluor 488 and goat anti-rat Alexa Fluor 555 and were purchased from Invitrogen. For flow cytometry, TLR4 (Sa15-21; Akashi et al., 2003) was conjugated to biotin and was a kind gift from Jonathan Kagan (Harvard Medical School, Boston, MA). Anti- strepavidin-APC was purchased from Biolegend.

Spleen-derived osteoclasts were cultured in 12-well tissue culture plates as described above. The plates were washed two times with cold PBS and lysed using RIPA buffer (0.1% triton X-100, 50 mM Tris, 300 mM NaCl, 5 mM EDTA), containing protease inhibitors (protease inhibitors cocktail P8340 (Sigma Aldrich) and 1 mM PMSF), and phosphatase inhibitors (Sigma Aldrich, P5726). Whole cell lysates were separated on 4–20% gradient TGX gels (BioRad), transferred to nitrocellulose membrane and probed for LC3B, p62, a3, LAMP2, mTOR, p-mTOR (S2448), p-Akt (S473), pan-AKT, p-p70S6K (T389) and actin (all from Cell Signaling Technologies (CST), except a3 (custom made^{15 (link)}) and LAMP2 (Developmental Studies Hybridoma Bank, Iowa, #ABL-93c)). Amersham ECL Prime Western blotting reagent was used as a detection reagent. Images were captured using BioRad ChemiDoc Gel Docking system.