Anti rfp

For ubiquitination assays, HEK 293 T cells were transfected with the indicated plasmids using Lipofectamine 2000 for 6 h. Then, the media were replaced with normal culture media. After transfecting for 24 h, the cells were treated with 10 µM MG132 for 2 h before harvesting. The cells were then lysed using co-IP lysis buffer supplemented with 20 mM NEM (Sigma, Burlington, MA). The cell lysates were centrifuged at 15,000 rpm at 4 °C for 20 min. Among the same amount of protein in the supernatant by by bicinchoninic acid (BCA) analysis, 10% supernatant was immunoblotted to confirm each protein expression. The remaining supernatants were incubated with 1 mg anti-GFP (Santacruz, CA, USA) or anti-RFP antibodies overnight at 4 °C. The antibody-binding proteins were pulled down by incubation for 1 h at 4 °C with 20 µL Protein A or G magnetic beads (ThermoFisher, Waltham, MA). The beads were washed three times with co-immunoprecipitation lysis buffer and heated in 1 × SDS sample loading buffer. The eluted proteins were immunoblotted with anti-RFP, anti-GFP, anti-ubiquitin (Santacruz, CA), or anti-β-actin antibodies.

IPA-3 (#3622) and PIR3.5 (#4212) were purchased from Tocris Bioscience and dissolved in sterile dimethylsulfoxide (DMSO) to make 50 mM stock solutions. Working solutions were prepared by 10 fold dilution of the stock solution in 50 mM Tris, pH 8.0, immediately before use. The cell density was adjusted to 3 × 10⁵ cells/ml for all experiments involving IPA-3 and PIR3.5 treatment. FRAX597 (#6029) was purchased from Tocris Biosciences and dissolved in sterile DMSO to make 10 mM stock solution. Working solution was prepared by dilution in cell culture medium.
Dasatinib was obtained from Selleckchem (#S1021), 200 µM stock solution was made in sterile DMSO. The antibodies against PAK1/2 are specified in Table 1. Other antibodies were purchased from the following providers: JMJD6 (sc 28348) and GFP (sc-9996) from Santa Cruz, β-actin (A5441) from Sigma-Aldrich, PAK3 from Cell Signaling (#2609). Anti-RFP was from Santa Cruz (sc-390909) or from Chromotek (6G6).

Fixed samples were decalcified in 20% EDTA (pH 7.5), embedded in paraffin and cut into 5 μm sections using an HM360 microtome (Microm). Hematoxylin (VWR) and eosin (Sigma–Aldrich) staining was performed to assess histomorphology. Tartrate-resistant acid phosphatase (TRAP) (Sigma) was used to detect osteoclasts according to the manufacturers’ protocols. For immunofluorescence staining, slides were subjected to sodium citrate buffer at 95 °C for 20 min for antigen retrieval, permeabilized with 0.5% Triton X-100 (Beyotime) for 10 min, and blocked with 5% BSA for 1 h. Slides were incubated with Anti-Runx2 (1:200, Abcam, ab23981), Anti-GFP (1:50, Santa Cruz, sc-9996), Anti-RFP (1:50, Santa Cruz, sc-390909), Anti-Klotho (1:100, R&D, AF1819), Anti-Klotho (1:100, Santa Cruz, sc-515939), or Anti-Rankl (1:100, R&D, AF462) antibody overnight at 4 °C, and a fluorescence-conjugated secondary antibody, Alexa Fluor 488 or 568 (Invitrogen, 1:1000) for 1 h at room temperature. Nuclei were counterstained with DAPI (Vector). Images were captured on an Olympus confocal microscope FV3000 (Olympus).