Figure 5. ZNRF1 mediates CAV1 polyubiquitination at lysine 39 and promote CAV1 degradation to modulate TLR4-mediated immune response.

(a) Schematic diagram of mouse CAV1 sequence and its 12 lysine residues, which were mutated to arginines. (b) HEK293T cells were co-transfected with V5-tagged single-lysine CAV1 mutants and either Flag-ZNRF1 or ZNRF1(C184A) as indicated. CAV1 ubiquitination was determined by immunoprecipitating V5-CAV1 and immunoblotting with anti-HA. (c) Immunoblot analysis of V5-tagged CAV1 in lysates from HEK293T cells expressing increasing amounts of ZNRF1 and either wild-type CAV1 or the CAV1(K39R) mutant as indicated. The intensities of the bands are shown as fold increases compared to those of control cells after normalization to GAPDH expression. (d) Immunoprecipitation of V5-tagged CAV1 proteins in lysates from HEK29T cells expressing Flag-ZNRF1 and either V5-CAV1 or CAV1(K39R), followed by immunoblotting with anti-HA antibody. (e,f) Cav1^−/− BMDMs were reconstituted with either V5-CAV1 or CAV1(K39R) and treated with LPS (100 ng ml⁻¹). (e) The mRNA expression levels of cytokines at 4 h post LPS were analysed by RT-qPCR, and (f) cytokine levels in supernatants were detected by ELISA at 12 h after LPS treatment. *P<0.05 (Student's t-test). The data are representative of three independent experiments performed in triplicate (error bars, s.d.).