Extended Data Fig. 6: CYLD inhibits ERK1/2 activity and their interaction with MEK1/2.

a, Knockout Cyld in MEFs increases ERK1/2 activity. Cyld^+/+ MEFs and Cyld^-/- MEFs were analyzed for ERK1 and ELK-1 phosphorylation and CYLD expression by Western blot. b, Interaction of endogenous CYLD and ERK1/2 in A375 cells was analyzed by co-IP with anti-ERK1/2 antibody. c, CYLD^4A has weakened ability to inactivate ERK1/2. A375 cells were infected with empty pCDH (EV), pCDH-CYLD, or pCDH-CYLD^4A lentiviral vector. Cell lysates were examined for ERK1/2 activation and CYLD/CYLD^4A expression. d, CYLD^4A is still able to deubiquitinate TRAF2. Myc-TRAF2 and HA-Ub were expressed in the presence or absence of Flag-CYLD^4A and Flag-CYLD^CA in HEK293T cells. Ubiquitination of Myc-TRAF2 was examined by d-IP with anti-Myc antibody. e, CYLD shows reduced interaction with ERK1^2DN. Flag-CYLD was incubated with immobilized GST, GST-ERK1, or GST-ERK1^2DN. The pulldown samples and input were analyzed by SDS-PAGE followed by Western blot and/or Ponceau S staining. f, Knockdown of CYLD increases ERK1-TRIM15 interaction. HEK293T cells were treated with NC or CYLD siRNA, and transfected with HA-TRIM15 alone or together with Flag-ERK1. Interaction between TRIM15 and ERK1 was assayed by co-IP with anti-Flag antibody. g, Knockout of CYLD increases TRIM15-ERK1/2 interaction. Cyld^+/+ and Cyld^-/- MEFs were analyzed for TRIM15-ERK1/2 interaction using co-IP with anti-ERK1/2 antibody. h, Interaction of Flag-ERK1 with endogenous MEK1 in HEK293T cells stably expressing shCtrl or shCYLD.

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