Journal: Cell communication and signaling : CCS

Article Title: The SIRT5-JIP4 interaction promotes osteoclastogenesis by modulating RANKL-induced signaling transduction.

doi: 10.1186/s12964-024-02021-x

Figure Lengend Snippet: Fig. 7 Identification of JIP4 as a target protein of SIRT5. A-B Protein samples from BMDMs treated with RANKL and M-CSF for 2 days were collected, and SIRT5 was immunoprecipitated (IP-SIRT5) for subsequent Western blot verification (A). The gel strip of the IP-SIRT5 sample was subjected to Coomassie Brilliant Blue staining, and the band between 25 kD and 35 kD, indicated by black boxes, represented the band of SIRT5 (B). The entire gel strip was further subjected to protein mass spectrometry analysis. C After excluding non-specifically bound proteins detected in the IgG negative control, subcellular localization of proteins detected in the IP-SIRT5 mass spectrometry was statistically analyzed and presented using a Venn diagram. The results show the quantity of proteins localized in the cytoplasm, mitochondria, nucleus, and other/unknown locations. D Representative immunofluorescence image of SIRT5 (green), the mitochondrial marker TOMM20 (red) and DAPI (blue) in BMDMs treated with MCSF or MCSF and RANKL for 1 day (scale bars, 20 μm/ 2 μm). E-F The line charts represent the fluorescence intensity of SIRT5 and TOMM20, presenting the distance from α to γ in BMDMs treated with MCSF (E) or MCSF and RANKL (F). G The top five cytoplasmic proteins ranked by protein scores in the IP-MS results were listed. H BMDMs were transfected with Sirt5-overexpressing lentivirus, treated with MCSF and RANKL for 2 days, and the protein levels of JIP4 and SIRT5 in the IP-SIRT5 products were detected

Article Snippet: Lysates were incubated with either IgG, SIRT5, or JIP4 antibody for 2 h and then with protein A/G-agarose beads (Santa Cruz, Dallas, USA) overnight at 4 °C.

Techniques: Immunoprecipitation, Western Blot, Stripping Membranes, Staining, Mass Spectrometry, Negative Control, Immunofluorescence, Marker, Fluorescence, Protein-Protein interactions, Transfection

Journal: Cell communication and signaling : CCS

Article Title: The SIRT5-JIP4 interaction promotes osteoclastogenesis by modulating RANKL-induced signaling transduction.

doi: 10.1186/s12964-024-02021-x

Figure Lengend Snippet: Fig. 8 SIRT5 promotes the phosphorylation of p38 and JNK through its interaction with JIP4. A Relative mRNA expression of JIP1, JIP2, JIP3, JIP4 in BMDMs after 2-day treatment of MCSF and RANKL (n = 3). B Relative mRNA expression of JIP4 and osteoclastogenesis-related genes including early osteoclastic differentiation markers (Nfatc1 and PU.1), osteoclast fusion markers (Dc-stamp and Oc-stamp), and osteoclast functional markers (Trap and Ctsk) (n = 3). C Expression levels of JIP4 and osteoclastogenesis-related proteins in CON, sh-JIP4 BMDMs after 4 days RANKL induction. D-E CON, sh-JIP4 BMDMs were induced with MCSF and RANKL for 5 days, and then TRAP staining was used to identify TRAP-positive multinucleated cells (D). Scale bar, 200 μm. The number of TRAP-positive multinucleated cells was calculated and presented graphically (n = 6) (E). F-G Images showing bone resorption areas in the CON and sh-JIP4 groups (F). Scale bar, 200 μm. The resorption pit areas were quantified. (n = 4) (G). H After exposing CON and sh-JIP4 BMDMs to RANKL for 0, 5 and 10 min, the phosphorylation levels of p38 and JNK were detected by Western blot. I After treating CON and sh-Sirt5 BMDMs with MCSF and RANKL for 2 days, protein levels of JIP4, p38, and SIRT5 in the IP-JIP4 products of BMDMs were examined. J After exposing CON and sh-Sirt5 BMDMs to RANKL for 0, 5 and 10 min, the phosphorylation levels of p38 and JNK were detected by Western blot. The data are presented as means ± SD. *p < 0.05, **p < 0.01 vs. CON, ***p < 0.001 vs. CON + RANKL

Article Snippet: Lysates were incubated with either IgG, SIRT5, or JIP4 antibody for 2 h and then with protein A/G-agarose beads (Santa Cruz, Dallas, USA) overnight at 4 °C.

Techniques: Phospho-proteomics, Expressing, Functional Assay, Staining, Western Blot