Pa5 29899

Whole-cell extracts (5~30 μg) were separated by 8–12% SDS-polyacrylamide gel electrophoresis, under reducing conditions and transferred onto nitrocellulose membranes (Amersham, Inc., Arlington Heights, IL, USA) by electroblotting. Protein transfer was verified using reversible staining with Ponceau S. Membranes were blocked with 3% non-fat dry milk in TBS-T (Tris-buffered saline and 0.2% Tween 20) for 1 h at room temperature. Proteins were detected using antibodies against vATPase E subunit (#PA5-29899; Thermo Fisher Scientific, Rockford, IL, USA), GPR120 (H-155; sc-99105; Santa Cruz Biotechnology, Dallas, TX, USA), GPR40 (SAB4501280, Sigma-Aldrich), aldolase A (sc-12059, Santa Cruz Biotechnology), Na⁺/K⁺-ATPase (sc-21712, Santa Cruz Biotechnology), and actin (sc-1615, Santa Cruz Biotechnology). Membranes were incubated overnight at 4 °C with primary antibodies diluted in TBS-T containing 3% non-fat dry milk. After washing with TBS-T, primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit, anti-mouse, or anti-goat) and visualized using an enhanced chemiluminescence detection system (Santa Cruz Biotechnology) after exposure to BioMax MR film (Kodak, Rochester, NY, USA). The target protein levels were compared to the levels of the loading control, actin.

Mature osteoclasts were fixed in ice-cold methanol or PBS containing 4% paraformaldehyde and stained with anti-vATPase subunit e1 (1:1000, PA5-29899, Thermo Fisher), anti-Cathepsin K (1:100, ab37259, Abcam), anti-Vinculin (1:100, V9264, Sigma), or control antibody (1:100, 5415 S, Cell Signaling), followed by fluorescently labeled secondary antibodies, following manufacturer’s instructions (Fisher). Some cells were also stained with Alexa Fluor 647-labeled Phalloidin (Thermo Fisher), following manufacturer’s instructions. The nuclei were stained with 1 µg/ml of Hoechst 33342 (Fisher) in PBS. Images were taken on an EVOS FL Auto (Thermo Fisher) and analyzed using the accompanying software. Because of intense TRAP staining in Elmo1^−/− osteoclast cultures, we counted the number of osteoclasts after cathepsin K staining by immunofluorescence. Cathepsin K-positive cells with three or more nuclei were counted using Image J. RosaYFP and Phalloidin stained osteoclast cultures were imaged on a Zeiss Imager Z2 with Apotome and analyzed using the AxioVision 4.8 software (Zeiss).