Fig 2: Ppp6r3 functional validation shows an effect of genotype on bone mass.(A) Schematic of the Ppp6r3 gene-trap allele (Ppp6r3tm1a(KOMP)Wtsi). Image obtained from the International Mouse Phenotype Consortium (IMPC). (B) Western blot of Ppp6r3 in experimental mouse spleens. Top left panel shows that PPP6R1 protein (control) levels are not affected by the Ppp6r3 gene-trap allele. Top right panel shows the effect of the gene-trap allele on PPP6R3 protein levels. The two bands are ostensibly due to different PPP6R3 isoforms. Bottom panel shows that PP6C protein (control) levels are not affected by the Ppp6r3 gene-trap allele. For raw blots, refer to Figure 4—source data 1. Least-squares means for spinal (C) and femoral (D) areal BMD (aBMD) dual X-ray absorptiometry (DXA) in Ppp6r3 wild-type (WT), heterozygous (HET), and mutant (MUT) mice. Contrast p values, adjusted for multiple comparisons are presented. *p ≤ 0.05. (E) Representative images of vertebrae for the Ppp6r3 experimental mice. Scale is shown on the bottom right. (F, G) Least-squares means for micro-computed tomography (μCT) measurements in the lumbar spines of Ppp6r3 WT, HET, and MUT mice. Contrast p values, adjusted for multiple comparisons are presented. *p ≤ 0.05. (H, I) Least-squares means for μCT measurements in the femoral midshaft of Ppp6r3 WT, HET, and MUT mice. Contrast p values, adjusted for multiple comparisons are presented. *p ≤ 0.05. Abbreviations: BV/TV – bone volume fraction, vBMD – volumetric bone mineral density, Tot.Ar – total area, Ma.Ar – medullary area. In panels (C, D and F–I), the center points represent the least-square means, and the error bars represent confidence intervals at a confidence level of 0.9. Data presented here are from both male and female mice. Figure 4—source data 1.Raw, unedited, PPP6R3 western blots.