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. 2011 Mar 15;124(6):921–932. doi: 10.1242/jcs.078055

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© 2011.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial Share Alike License, which permits unrestricted non-commercial use, distribution and reproduction in any medium provided that the original work is properly cited and all further distributions of the work or adaptation are subject to the same Creative Commons License terms.

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Fig. 6. — RNF41 blocks LIFRα and IL-6Rα cleavage by cathepsin L and enhances their shedding. (A,B) Chloroquine stabilises C-terminal LIFR and IL-6Rα fragments, which are blocked by cathepsin L inhibition (left panels) or RNF41 expression (right panels). HEK293T cells transiently transfected with C-terminal HA-tagged human LIFRα (LIFRα–HA) (A) or IL-6Rα (IL-6Rα–HA) (B) were left untreated (DMSO) or were incubated overnight solely with chloroquine or together with the indicated protease inhibitors (left panels); or were cotransfected with a full-length Etag–RNF41 or Etag–RNF41 ΔRING construct and left untreated or incubated with chloroquine (right). Cell lysates were analysed by western blotting (WB). FL, full length; *, intermediate IL-6Rα cleavage product; CTS, C-terminal stub. (C,D) RNF41 enhances LIFRα (C) and IL-6Rα (D) shedding. Cell media supernatants from the transfectants in the right panels of Fig. 6A,B were analysed for soluble LIFRα and IL-6Rα levels. E-tagged RNF41 or RNF41 ΔRING expression was verified by western blotting. (E,F) RNF41-enhanced LIFRα (E) and IL-6Rα (F) shedding is reversed by metalloprotease inhibitors. HEK293T cells transfected with hLIFRα–HA or hIL-6Rα–HA with or without full-length Etag–RNF41 were incubated overnight in starvation medium with TAPI-1 or GM6001 and soluble receptor levels in the cell medium supernatants were quantified. E-tagged RNF41 expression was verified by western blotting. (G) RNF41-enhanced IL-6R shedding is reversed by ADAM10 silencing. HEK293T cells, reverse transfected with siRNA targeting ADAM10 or ADAM17, were cotransfected the next day with hIL-6Rα–HA with or without full-length Etag–RNF41 and soluble IL-6Rα levels in the cell medium supernatants were quantified. Values are means ± s.d. (n=3). *P<0.01 or **P<0.05 (Student's t-test) compared with – RNF41; ^§P<0.01 or ^§§P<0.05 (Student's t-test) compared with +RNF41.