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. Author manuscript; available in PMC: 2017 Oct 10.
Published in final edited form as: Cancer Cell. 2016 Oct 10;30(4):595–609. doi: 10.1016/j.ccell.2016.09.004

Figure 6. p62 regulates the formation of the VDR:RXRα dimer in response to calcipotriol.

Figure 6

(A and B) Endogenous interaction of RXRα (A) or VDR (B) with p62 in response to calcipotriol (Cal). Cell lysates and immunoprecipitates were analyzed for the levels of specified proteins; se: short exposure; le: long exposure. (C) Proximity ligation assay (PLA) of RXRα–p62 and VDR– p62 interactions (red) in WT HSC treated with vehicle (DMSO) or 100 nM Cal for 24 hr. (D) Cell lysates and V5-tagged immunoprecipitates of HEK293T cells transfected with the indicated cDNAs in expression vectors were analyzed by immunoblotting. (E) Recombinant His-VDR was incubated with FLAG-RXRα in the presence of recombinant MBP-p62 in response to Cal and interactions were analyzed by immunoblot in His-beads pulldown. (F) Cell lysates and endogenous VDR-immunoprecipitates of WT and p62KO HSC treated with or without Cal were analyzed by immunoblotting. (G) PLA of VDR:RXRα heterodimer (red) in WT and p62KO HSC treated with vehicle (DMSO) or 100 nM Cal for 24 hr. (H) Cell lysates and V5-tagged immunoprecipitates of HEK293T cells transfected with the indicated cDNAs in expression vectors were analyzed by immunoblotting. (I) PLA of SRC-1:VDR (red) in WT and p62KO HSC treated with vehicle (DMSO) or 100 nM Cal for 24 hr. (J) Schematic representation of domain structure of p62 and a summary of the interactions of RXRα with the different domains of p62. (K) Schematic representation of domain structure of RXRα and a summary of the interactions of p62 with the different domains of RXRα. (L) Cell lysates and V5-tagged immunoprecipitates of HEK293T cells transfected with the indicated cDNAs in expression vectors were analyzed by immunoblotting. (M) Immunoblot analysis of αSMA, p62 and Actin in WT, p62KO and p62KO HSC reconstituted with the indicated constructs and treated with vehicle (DMSO) or Cal for 24 hr. (N) qPCR analysis of mRNA of Mmp13, Cyp24a1, and Acta2 in the reconstitution experiment as in (M). (O) Model for the role of p62 in the regulation of VDR:RXR heterodimerization. Scale bars, 20 μm. Results are presented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.