Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Phosphorylation-mediated self-ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) by mammalian Sterile 20-like kinase 4 (MST4). (A) Western blot analysis of phosphorylation of TRAF6, downstream nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, osteoclast markers proto-oncogene c-Fos (c-Fos), nuclear factor of activated T-cells (NFATc1) expression levels, and statistical graphs. (B) Immunoblot analysis of TRAF6 phosphorylation at 0, 10, 20, and 30 min post-receptor activator of nuclear factor κB ligand (RANKL) stimulation. (C) Immunoblot analysis of serine (Ser) and threonine (Thr) phosphorylation in TRAF6 after 30 min of RANKL stimulation, and immunoblot analysis of TRAF6 ubiquitination after MST4 silencing or overexpression. (D) In vitro ubiquitination assay evaluating the processing of purified TRAF6 containing histidine tags in the absence of MST4, wild-type MST4 (WT), or kinase-inactive MST4 mutant (KR), along with purified ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2 (E1-E2) and ubiquitin, assessed by immunoblot using anti-ubiquitin (Ub) and anti-TRAF6 antibodies after immunoprecipitation with anti-TRAF6 antibody, with detection of MST4 by Ponceau staining. (E) Kinase assay evaluating the effect of WT and KR on TRAF6-C as a substrate, assessed by autoradiography and Coomassie blue staining. ∗ P < 0.05 between groups, with all cell experiments repeated thrice. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; p-S/T: phospho-serine/threonine; p-MAPK: phospho-mitogen-activated protein kinase; Autorad: autoradiography; CBB: Coomassie brilliant blue staining.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Sterility, Western Blot, Protein-Protein interactions, Expressing, Over Expression, In Vitro, Purification, Mutagenesis, Immunoprecipitation, Staining, Kinase Assay, Autoradiography

Journal: Journal of Pharmaceutical Analysis

Article Title: MST4 as a key driver of osteoclast activation in osteoporosis

doi: 10.1016/j.jpha.2025.101401

Figure Lengend Snippet: Impact of tumor necrosis factor receptor-associated factor 6 (TRAF6) phosphorylation site mutations Thr463 and Thr486 on osteoclast differentiation. (A) Western blot analysis of downstream nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, osteoclast markers cellular homolog of the Finkel–Biskis–Jinkins murine osteosarcoma viral oncogene (c-Fos), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) expression levels, and statistical charts. (B) Representative images and statistical analysis of tartrate-resistant acid phosphatase (TRAP) staining. (C) Representative images and statistical analysis of tartrate-resistant acid phosphatase (TRAcP) staining. (D) Representative images and statistical analysis of resorption pits on hydroxyapatite-coated Corning Osteo Assay plate. ∗ P < 0.05 compared between groups, with all cell experiments repeated thrice. GAPDH: glyceraldehyde 3-phosphate dehydrogenase; F-actin: filamentous actin.

Article Snippet: The proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane (Cat. No. F619534-0005, Sangon Biotech Co., Ltd., Shanghai, China), which was blocked with 5% non-fat milk (Cat. No. A600669, Sangon Biotech Co., Ltd., Shanghai, China) at room temperature for 1 h. The membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: MST4 (Cat. No. ab52491, 1:100,000; Abcam, Cambridge, UK), proto-oncogene c-Fos (Cat. No. ab208942, 1:1000; c-Fos, Abcam, Cambridge, UK), nuclear factor of activated T-cells, cytoplasmic 1 (Cat. No. ab25916, 1:1000; NFATc1, Abcam, Cambridge, UK), tartrate-resistant acid phosphatase (TRAP; Cat. No. A13002, 1:1000; Abclonal, Wuhan, China), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; Cat. No. #8242, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-NF-κB (Cat. No. #3033, 1:1000;, Cell Signaling Technology, Danvers, MA, USA), mitogen-activated protein kinase (MAPK; Cat. No. #4695, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-MAPK (Cat. No. #4370, 1:1000; Cell Signaling Technology, Danvers, MA, USA), p-S/T (Cat. No. 05–1923, 1:200; Merck, Darmstadt, Germany), with GAPDH (Cat. No. ab181602, 1:10,000; Abcam, Cambridge, UK) as the internal control.

Techniques: Phospho-proteomics, Western Blot, Protein-Protein interactions, Expressing, Staining