The lack of expression of functional LRP5 protein was confirmed by immunnoblotting.
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).
Protein from rat tail biopsies was isolated in lysis buffer (50 mM Na 2 HPO 4 , 1 mM sodium pyrophosphate, 20 mM NaF, 2 mM EDTA, 2 mM EGTA, 1% Triton X-100, 0.5 mM DTT, protease inhibitor tablet Roche, Basal, Switzerland) using lysing matrix M (MP Biomedicals, Irvine, CA) for homogenization in the FastPrep-24 sample disruption instrument (MP Biomedicals). The samples were centrifuged at 21,000 x g for 30 min at 4 °C to pellet nonlysed tissue. We analyzed 40 μg of protein by SDS-polyacrylamide gel electrophoresis on an 8% Trisglycine gel (Thermo Fisher Scientific) and transferred to PVDF western blotting membrane overnight at 4 °C. Immunoblotting was performed using the following antibodies: Lrp5 (D80F2) rabbit mAb (Cell Signaling, 5731), β-actin (13E5) rabbit mAb (Cell Signaling, 5125) (Cell Signalling Technology, Danvers, MA), and V5 Tag mAb, HRPP (Thermo Fisher Scientific, R96125).