Fig. 6: Hsp90ab1 mediates the signal transmission from B7-1 to the LRP5/β-catenin pathway.

A, B Graphic presentation showing the predicted binding sites between B7-1 and Hsp90ab1 residues L65 (▴1), K69 (▴2), R168 (▴3), D170 (▴4). The full structure of B7-1 contains IgC domain, IgV domain, transmembrane domain (TM) and Tail. The full structure of Hsp90ab1 contains N-terminal domain (NTD), middle domain (MD), C-terminal domain (CTD), and marked with ▴ for specific point mutation. C, D Representative immunoblotting and quantitative data showing the specific binding site in Hsp90ab1 for interaction with B7-1 is ▴2 (K69). 293 T cells were transfected with B7-1 expression plasmid (Flag tagged), and co-transfected with HA tagged Hsp90ab1 wildtype plasmid (Full) or mutation plasmids for 24 hours. Whole cell lysates were immunoprecipitated with a HA-tag antibody and immunoblotted with B7-1 or HA antibody. Total diluted lysates were used as input. Each dot on the bar plot indicated an individual repeated experiment run in duplicate. *P < 0.05 versus the wildtype group. E, F Representative Western blot and quantitative data of active β-catenin, Zo-1 and Podocalyxin (podx) in indicated group. MPC5 cells were transfected with siHsp90ab1 for 24 h, followed by transfection with pFlag-B7-1 plasmid and Hsp90ab1 wildtype plasmid (Full) or K69A mutated (▴2) plasmid for another 24 hours. *P < 0.05 versus controls, ^#P < 0.05 versus pFlag-B7-1 group, ^†P < 0.05versus cells co-transfected with Hsp90ab1 wild type plasmid and pFlag-B7-1 plasmid. G Micrographs showing the expression of Zo-1 in MPC5 cells pretreated with siHsp90ab1, and co-transfected with pFlag-B7-1 and wild type plasmid of Hsp90ab1 (Full) or mutated plasmid (▴2). Arrow indicates the positive staining. Bar = 25 μm. H Molecular structure of LRP5 from homology modeling and putative binding model of B7-1, LRP5 and Hsp90ab1 dimer. I, J Representative immunoblotting show the interaction between LRP5 and B7-1 or Hsp90ab1 in cells or kidney tissue lysates as indicated. The protein lysates were immunoprecipitated with antibodies against B7-1 and Hsp90ab1 respectively, and blotted with anti-LRP5. K, L Western blot and quantification of LRP5 in 6-month-old WT and Tg mice. **P < 0.01 versus WT group. n = 5. M Representative micrographs showing the expression of LRP5 and Flag-tag (B7-1) in WT and Tg mice. Arrows indicate the specific colocalization in podocytes. Bar = 25 μm. N, O Western blot and quantification of active β-catenin and Synaptopodin. MPC5 cells were transfected with pFlag-B7-1 plasmid (or empty vector), and co-transfected with siRNA to Hsp90ab1 (siHsp90ab1) or LRP5 (siLRP5). *P < 0.05, **P < 0.01 versus B7-1 overexpression group. n = 3^. The full length original western blots for all results are provided in Supplementary File 1.