Fig. 5

LRP5 interacts with TβRs and stimulates the formation of TβRI/TβRII heterodimers. a Immunostaining of LRP5 (green color; scale bar = 25 μm) and TβRI (red color; scale bar = 25 μm) in the kidney sections from WT mice at day 5 post-surgery showing co-localization of LRP5 and TβRI in the injured tubules of UUO kidneys (n = 5–7). b IP of endogenous LRP5 in HKC-8 cells. LRP5 was precipitated with its specific antibody. The precipitated proteins were immunoblotted for TβRI, TβRII, and LRP5. c, d IP of (c) TβRI and (d) LRP5 in HEK293A cells co-transfected with TβRI-His and LRP5-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β1 for 15 min. TβRI-His was precipitated by Ni-NTA resin and LRP5-Flag by anti-Flag resin. The precipitated proteins were immunoblotted for His and Flag. e, f IP of (e) LRP5 or (f) TβRII in HKC-8 cells co-transfected with TβRII and LRP5 for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRII and LRP5 were precipitated using their specific antibodies. The precipitated proteins were immunoblotted for TβRII and LRP5. g IP of TβRI in HEK293A cells co-transfected with TβRI-His and LRP5FL-Flag, LRP5∆N-Flag or LRP5∆C-Flag for 48 h. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His and Flag. h Measurement of 3TP-Lux luciferase activity in HKC-8 cells after transfection of the 3TP-Lux plasmid, a renilla plasmid and a control plasmid or plasmid expressing LRP5FL, LRP5∆N, or LRP5∆C for 48 h, followed by the treatment with/without 4 ng/ml TGF-β1 for 16 h. Relative luciferase activity was presented as folds of that in the cells with transfection of pcDNA3 control (n = 3). i IP of TβRI in HKC-8 cells co-transfected with TβRI-His and LRP5-Flag or LDLR-Flag for 48 h, followed by the treatment with/without 2 ng/ml TGF-β for 15 min. TβRI-His was precipitated by Ni-NTA resin. The precipitated proteins were immunoblotted for His, Flag, and TβRII. Arrowheads indicate non-specific bands; arrows indicate target proteins. All values are expressed as mean ± SEM. **p < 0.01; n.s., not significant, by two-way ANOVA with pair-wise multiple comparisons