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. 2017 Jun 8;8:15502. doi: 10.1038/ncomms15502

Figure 4. ZNRF1 interacts with CAV1 and promotes CAV1 ubiquitination in response to LPS.

Figure 4

(a) HEK293T cells were co-transfected with V5-tagged CAV1 and Flag-tagged ZNRF1 as indicated. Interactions between ZNRF1 and CAV1 were detected by immunoprecipitation followed by immunoblot analysis with the indicated antibodies. (b) Immunoprecipitation of ZNRF1 from lysates of RAW264.7 cells treated with LPS (100 ng ml−1) for the indicated times, followed by immunoblot analysis with the indicated antibodies. The intensities of the bands are shown as fold increases compared to those of untreated cells after normalization to ZNRF1 level. (c) Schematic diagram of full-length CAV1 and six truncated forms of CAV1 with a C-terminal V5 tag. (d) HEK293T cells were co-transfected with Flag-tagged ZNRF1 and V5-tagged full-length or truncated CAV1 as indicated, and interactions between CAV1 and ZNRF1 were identified by immunoprecipitation and immunoblotting with the indicated antibodies. (e) Schematic diagram of full-length ZNRF1 and two truncated forms of ZNRF1 with a C-terminal Flag-tag. (f) HEK293T cells were co-transfected with V5-tagged CAV1 and Flag-tagged full-length or truncated form of ZNRF1 as indicated, and interactions between CAV1 and ZNRF1 were identified by immunoprecipitation and immunoblotting with the indicated antibodies. (g) HEK293T cells were co-transfected with Flag-tagged ZNRF1 or ZNRF1(C184A) mutant and V5-tagged CAV1 and HA-tagged ubiquitin. CAV1 ubiquitination was determined by immunoprecipitating V5-tagged CAV1 and subsequent immunoblotting with anti-HA. (h) In vitro ubiquitination assays were carried out with bacterially expressed His-CAV1, ZNRF1 or ZNRF1(C184A), purified ubiquitin catalytic components as indicated. The mixtures were then subjected to immunoblotting with the indicated antibodies. Arrows indicate Ubiquitin-conjugated CAV1. (i,j) RAW264.7 cells transduced with lentiviruses expressing shScr or shZnrf1 and BMDMs from Znrf1F/F and Znrf1Δ mice were treated with LPS (100 ng ml−1) for the indicated times after MG132 pre-treatment. CAV1 ubiquitination was determined by immunoprecipitating CAV1 and subsequent immunoblotting with anti-ubiquitin antibody. The data are representative of three independent experiments performed in triplicate.