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. 2018 Mar 7;25(11):2010–2022. doi: 10.1038/s41418-018-0090-y

Fig. 5.

Fig. 5

HACE1 interacts with and promotes fibronectin ubiquitination. a GST or GST-HACE1 was incubated with reconstituted fibronectin (FN) or vitronectin (VN). Bound proteins were analysed by western blotting using antibodies against FN or VN. b Purified FN (1 µM) was incubated with ATP, E2 ligase, ubiquitin and recombinant HACE1 (0.4 µM) as indicated and then analysed by western blotting using an antibody against FN. c COS cells were transfected with an empty vector or vectors encoding HA-tag-K27 and control (siCtl) or HACE1 (siH1) siRNA. Total cell extracts (CL) were analysed by western blotting using antibodies against FN, HACE1, HA or HSP90 (loading control). The secreted proteins (CM) were analysed by western blotting using an antibody against FN. d Cellular proteins from cells treated as in C were subjected to immunoprecipitation with a FN antibody and analysed by western blotting using antibodies against HA-tag or FN. e Cells were transfected with an empty vector (EV) or plasmid encoding for HA-tagged K27 ubiquitin (K27-HA) or Flag-tagged K27R ubiquitin (K27R-Flag). Cell lysates were analysed by western blotting using antibodies against Flag, HA or K27 ubiquitin. f The cell lysates (CL) as in (e) were analysed by western blotting using antibodies against FN or HSP90 (loading control). Conditioned media (CM) from the same cells were analysed by western blotting using antibodies against FN or VN (loading control)