Atp6v1a

RAW264.7 cells (ATCC) were cultured in 10% FBS in DMEM (Gibco/Life Technologies) containing 10 µg/ml penicillin/streptomycin. For RAW-derived osteoclasts, RAW cells were cultured in a presence of 200 ng/ml RANKL for 5 days. For fractionation experiments, RAW-derived osteoclasts or RAW cells were cultured in 100 mm dishes, washed with 5 ml of cold PBS twice, and then scraped in 380 µl of homogenization Buffer (205 mM sucrose, 1 mM EDTA and 10 mM HEPES, pH 7.4) per dish (4 × 100 mm dishes/group). For each experimental group, combined cell suspension was homogenized using Dounce homogenizer (tight) with 60 strokes. The homogenate was cleared (from the nuclei and intact cells) by centrifugation at 4 °C, 500 × g for 10 min. After clearing, the homogenate was centrifuged at 100,000 × g for 30 min at 4 °C. Supernatant was removed and represented a cytosolic fraction, while the pellet was re-suspended in homogenization buffer and represented a membrane fraction. SDS was added to the both fractions to a final concentration of 1%. Protein concentration was measured using Bradford-based method (BioRad protein assay reagent, #500–006). Samples (10 µg) from each fraction separated on 4–20% SDS PAGE and transferred to the nitrocellulose membrane. Immunoblotting was performed as described above using mTOR, ATP6V1A (GeneTex), a3, LAMP2, Raptor (CST, #2280), and LAMTOR1 (CST, #8975) antibodies.