Fig. S4.
Localization, abundance, and interactions of components of the Cop1 complex and activity of the core Cullin complex are unchanged by ERK activity. (A) Immunoblots of E1A/Hras-transformed wild-type or Det13×f/3×f MEFs that were treated with 1 µM MEK inhibitor (MEKI) for 1 h. 3×Flag-DET1 was immunoprecipitated (IP) with anti-Flag beads where indicated. (B) Immunoblots of 293.Kras cells transfected with Flag-ETV5 were treated with 100 ng/mL doxycycline (Dox) overnight, then with 1 µM MLN4924 for 1 h, and then with 1 µM MEK inhibitor for another 1 h. Flag-ETV5 was immunoprecipitated with anti-Flag beads where indicated. (C) Immunoblots of E1A/Hras-transformed MEFs that were treated with 1 µM MEK inhibitor for 1 h and then were fractionated into cytosol (Cyto) and nuclei (Nucl). WCL, whole-cell lysate starting material. (D) Immunoblots of E1A/Hras-transformed MEFs treated with DMSO or 1 µM ERK inhibitor (ERKI) for 1 h and fractionated through a Superose 6 10/300 GL column. (E) Immunoblots of COP1-containing complexes affinity purified from Cop1fh/fh MEFs treated with 1 µM ERK inhibitor or DMSO for 1 h. Flag.HA-COP1 was immunoprecipitated with anti-Flag beads and eluted with 3×Flag peptide. The eluates were fractionated through a Superose 6 10/300 GL column and concentrated by acetone precipitation for Western blot analysis. (F) Immunoblots of 293.Kras cells treated with doxycycline overnight and then with MEK inhibitor or MLN4924 for 1 h before UV irradiation. CDT1, chromatin licensing and DNA replication factor 1; CDT2, chromatin licensing and DNA replication factor 2; DDB2, damage-specific DNA-binding protein 2; NED, Nedd8. Each small blue ball represents a ubiquitin in a polyubiquitin chain.