Fig 1: Gene expression profile of the six selected genes. We investigated the relative gene expression profile of the six genes with an AS event significantly associated with PDB (CASC4, LGALS8, PIDD, RHOT1, TBC1D25, USP4) in OC cultures from 22 PDB patients (including 10 PDBP392L) and 22 healthy donors (HD) (including 10 HDP392L). Total RNA extraction was performed on cultures from PBMC-derived OCs, followed by real-time PCR experiments. Relative levels were normalized with respect to a set of three reference genes. We compared the mean, normalized, relative expression between the four groups using Student’s t-test. Differential expression is reported as the log2 ratio. Comparisons between all PBD and HDwt, PBDP392L and PBDwt as well as HDP392L and HDwt are presented. *p < 0.05, **p < 0.01.
Fig 2: Western blot analysis of PIDD , TBC1D25 , RHOT1 , USP4 and LGALS8 encoded proteins in pagetic or control OCs. The protein expression of the candidate genes was analyzed in OC cultures derived from PDB or HD without any p62 mutations. A- Western blot analyses of each encoded protein were performed using antibodies directed against PIDD, TBC1D25, RHOT1, USP4 and LGALS8 proteins or actin. B- Optical densities for bands corresponding to each protein were corrected with the optical density obtained for bands corresponding to actin, and computed in graphical representations (n = 4 to 6 in each group, in 2 independant experiments). Analyses are reported as mean ratio ± SD (*p = 0.05, ** p = 0.01).
Fig 3: Immunofluorescent analysis of TBC1D25 , USP4 and PIDD encoded proteins in mature human OCs. Immunofluorescence tests were performed on mature osteoclasts derived from CBMs, using antibodies directed against each protein (encoded by TBC1D25, USP4 and PIDD), and fluorescent secondary antibodies as well as Alexa Fluor 633-conjugated phalloidin. Nuclei were stained with DAPI. Images are representative of three independent experiments. Scale bar represents 10 µm.
Fig 4: Western blot analysis of PIDD , TBC1D25 , RHOT1 , USP4 and LGALS8 encoded proteins. The expression of PIDD, TBC1D25, RHOT1, USP4 and LGALS8 was analyzed by western blot of a protein extract of OC cultures derived from CBMs, and from 293 T cells. ß-actin was used as an internal control. 293 T cells were transfected with a scrambled control siRNA (scramble), or with an siRNA specific for each gene (regions common to all isoforms were targeted). Western blot analyses of each encoded protein were performed 72 to 96 hours post siRNA transfection in 293 T cultures, and in OC cultures, with antibodies directed against PIDD, TBC1D25, RHOT1, USP4 and LGALS8 proteins or actin. Images are representative of three independent experiments. Two different gels are shown for the analysis in OCs. Putative isoforms are indicated to the left of each gel image (numbering from Table 3).
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