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. Author manuscript; available in PMC: 2022 Mar 8.
Published in final edited form as: Nat Cell Biol. 2021 Sep 8;23(9):978–991. doi: 10.1038/s41556-021-00732-8

Extended Data Fig. 6. CYLD inhibitsERK1/2 activity and their interaction with MEK1/2.

Extended Data Fig. 6

a, Knockout Cyldin 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, CYLD4Ahas weakened ability to inactivate ERK1/2. A375 cells were infected with empty pCDH (EV), pCDH-CYLD, or pCDH-CYLD4A lentiviral vector.Cell lysates were examined for ERK1/2 activation and CYLD/CYLD4Aexpression.d, CYLD4Ais still able to deubiquitinate TRAF2. Myc-TRAF2 and HA-Ub were expressed in the presence or absence of Flag-CYLD4Aand Flag-CYLDCA in HEK293T cells. Ubiquitination of Myc-TRAF2 was examined by d-IP with anti-Myc antibody.e, CYLD shows reduced interaction with ERK12DN. Flag-CYLD was incubated with immobilized GST, GST-ERK1, or GST-ERK12DN. 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.