Identification of the aa 191-230 region is important for EKLF stability. (A) Multiple amino acid sequence alignment of the aa 191-230 region (in red box) on EKLF proteins from the indicated species using the Clustal X 2.0 program. (B) HEK293T cells transfected with Myc-EKLF or Myc-EKLF-Δ191-230 were treated with CHX for indicated times and then analyzed by western blot. Representative western blot and quantification of relative protein levels are shown. (C) HEK293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-EKLF, Myc-EKLF-Δ191-230, and HA-Ub. Before collection, cells were treated with MG132 (20 μM) for 6 hours. Immunoblot analysis of EKLF ubiquitination (detected by EKLF antibody) in cell lysates immunoprecipitated with HA antibody. (D) HEK293T cells were transfected with various combinations of plasmids as indicated below each column. The relative luciferase activities were measured after 48 hours by a dual-luciferase reporter assay. All activities were normalized to pGL3-Basic activity. (E) HEK293T cells transfected with Myc-EKLF or Myc-EKLF (Gln211Arg) were treated with CHX for indicated times and then analyzed by western blot. Representative western blot and quantification of relative protein levels are shown. (F) HEK293T cells were transfected with Flag-GPS2 together with Myc-EKLF or Myc-EKLF (Gln211Arg). Cell lysates were pulled down with anti-c-Myc agarose and subjected to immunoblot with Myc or Flag antibody. (G) HEK293T cells transfected with Myc-EKLF or Myc-EKLF (Gln211Arg) together with Flag-GPS2 or control vector were treated with CHX for indicated times and then analyzed by western blot. Representative western blot and quantification of relative protein levels are shown. Data are representative of 3 independent experiments (C,F). Values are mean ± SEM (n = 3 replicates). ***P < .001; 2-tailed unpaired t test (D) or 2-way ANOVA test (B,E,G).