Sp 61

Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Shanghai, China) containing protease inhibitors and phosphatase inhibitors (C0001 and C0004, TargetMol, Massachusetts, USA). Total lysates were separated by sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE, C671102, Sangon Biotech) and transferred to polyvinylidene fluoride (PVDF) membranes (IPVH00005, Millipore, Burlington, MA, USA). The membranes were incubated with GMFB-specific antibodies (1:200, SP-61, Santa Cruz) and visualized with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). The corresponding bands were detected using an enhanced chemiluminescent (ECL) detection kit (36208ES60, Yeasen).

For immunocytochemical staining, the fixed mature osteoclasts were incubated with primary antibodies against Arp2 (1:50, D221703, BBI, Sangon Biotech, Shanghai, China), GMFB (1:50, SP-61, Santa Cruz, CA, USA), DBP (1:150, D163666, BBI), DNase I (1:100, D222246, BBI), or MKL1 (1:50, sc-398675, Santa Cruz) at 4 °C overnight, followed by Alexa 647- and Alexa 488-conjugated secondary Abs (1:1000, abcam, Cambridge, UK) for 1 h. Images were taken on a Leica TCS SP5 confocal scanning laser microscope (Leica Laser Technik, Solms, Germany).