Fig. 1: E3 ligase CHFR interaction with VE-cadherin (CDH5) and ubiquitylation of VE-cadherin. | Nature Communications

Fig. 1: E3 ligase CHFR interaction with VE-cadherin (CDH5) and ubiquitylation of VE-cadherin.

From: Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions

Fig. 1

a Schematic of work flow determining the binding of soluble C-terminal green fluorescent protein (GFP)-fused hVE-cadherin to human recombinant proteins. b Volcano plot showing binding of hVE-cadherin-GFP (mean fluorescence intensity vs. p-value) to proteins in the microarray. Red dots show positive values and blue dots show negative values. c Scattered plot identifying the top 9 ubiquitin E3 ligases binding to hVE-cadherin. b, c shown are mean values (unpaired Student’s t test). d Schematics of domain structures of human wildtype (WT) CHFR and CHFR mutants lacking forkhead-associated domain (ΔFHA-CHFR), RING finger domain (ΔRF-CHFR), cysteine-rich domain (ΔCR-CHFR), or poly-ADP ribose binding zinc-finger domain (ΔPBZ-CHFR). N-terminal eGFP-tagged WT-CHFR, ΔFHA-CHFR, ΔRF-CHFR, ΔCR-CHFR, and ΔPBZ-CHFR were generated in a pEGFP-C2 expression vector for this study. e Human dermal microvascular endothelial cells (HMEC, an endothelial cell line) were transfected with N-terminal GFP tagged WT-CHFR, ΔFHA-CHFR, ΔRF-CHFR, ΔCR-CHFR, or ΔPBZ-CHFR (1.5 μg/ml). The cells were incubated at 48 h with MG132 (10 μM) and the lysates were used for immunoblot (IB) analysis. f Transfected HMEC were used for anti-GFP-agarose beads pull-down assays to study interactions of WT CHFR and CHFR mutants with VE-cadherin. Bottom panel shows quantification of CHFR binding to VE-cadherin as ratio of VE-cadherin (VE-cad)-to-GFP-CHFR. arb. units, arbitrary units. g HEK293 cells transfected with HA-tagged ubiquitin (HA-Ub) (0.5 μg/ml) alone or co-transfected with WT-CHFR (1.5 μg/ml), ΔFHA-CHFR (1.5 μg/ml), or ΔRF-CHFR (1.5 μg/ml) were used to study ubiquintylation of VE-cadherin. The cells at 24 h were infected with recombinant adenovirus expressing C-terminal GFP-tagged mVE-cadherin (5 pfu/cell). At 24 h thereafter the cells were pretreated with MG132 (10 μM) for 4 h and cell lysates were used for IB analysis. In (h), the cell lysates were immunoprecipitated with anti-VE-cadherin antibody and blotted with antibody specific to K48-linked or K63-linked poly-Ub. Blots were re-probed with antibody specific to either VE-cadherin or CHFR. Representative results from two independent experiments are shown.

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