Extended Data Fig. 3: ArpC4 is a bona fide substrate of UFMylation.
From: Akt-phosphorylated UFL1 UFMylates ArpC4 to promote metastasis
(a) Lysates from HEK293T cells expressing Flag-UFL1 alone or together with the indicated HA-tagged each single subunit of the Arp2/3 complex were immunoprecipitated with anti-Flag antibody, followed by western blot analysis. (b) Lysates from HEK293T cells expressing HA-UFL1 alone or HA-UFL1 plus Flag-tagged Arp3, Cortactin, Coronin, and Cofilin as indicated were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. (c) Lysates from HEK293T cells expressing HA-UFL1 alone or HA-UFL1 plus Flag-tagged N-WASP were immunoprecipitated by anti-Flag antibody, followed by western blot analysis. (d) Myc-tagged UBA5 and UFC1, HA-tagged UFL1, UFBP1, and UFM1-ΔC2 were expressed in HEK293T cells with Flag-tagged ArpC4 as indicated. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (e) ArpC4, UFC1, UFL1, and UFBP1, together with UFM1-ΔC2 or UFM1-ΔC3, were expressed in HEK293T cells. Denatured cell lysates were subjected to pull-down with Ni-NTA resins, followed by western blot analysis. (f) Flag-tagged ArpC4 and the UFMylation components were expressed in HEK293T cells with or without HA-tagged UFSP2. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (g) His-tagged wild-type ArpC4 or its KO mutant (all lysine residues were replaced with arginine) was expressed in HEK293T cells with the UFMylation components as indicated. Denatured cell lysates were subjected to pull-down with Ni-NTA resins, followed by western blot analysis. (h) UFSP2-KO H1299 cells expression control or Flag-UFM1-ΔC2 were infected with lentiviruses expressing either control shRNA or ArpC4 shRNA as indicated. Denatured cell lysates were subjected to pull-down with anti-Flag antibody, followed by western blot analysis. (i) Coomassie blue staining of recombinant His-tagged UBA5, UFC1, UFM1-ΔC2, UFL1/UFBP1, and MBP-tagged ArpC4. All experiments were repeated three times independently with similar results. The shown blots/gels are representative of three independent experiments.
