Figure 5.
GPS2 increases protein stability of EKLF but does not alter its ubiquitination. (A-B) Immunoblot analysis of GPS2 and EKLF protein levels in GPS2-knockdown and control MEL cells (A), as well as in Gps2+/+ and Gps2−/− E12.5 fetal livers (B). (C-D) Real-time PCR analysis of GPS2 and EKLF mRNA levels in GPS2-knockdown and control MEL cells (C), as well as in Gps2+/+ and Gps2−/− E12.5 fetal livers (D). (E) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2 or control vector were treated with CHX for indicated times and then analyzed by western blot. (F-G) MEL cells with/without stable overexpression of GPS2 (F) or knockdown of GPS2 (G) were treated with CHX for indicated times and then analyzed by western blot. (H) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2-Δ111-150 or control vector were treated with CHX for indicated times and then analyzed by western blot. (I) HEK293T cells transfected with Myc-EKLF-Δ191-230 together with Flag-GPS2 or control vector were treated with CHX for indicated times and then analyzed by immunoblot. (J) HEK293T cells transfected with Myc-EKLF-Δ1-68 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. (K) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2 or control vector were treated with vehicle, MG132 (20 μM), or E64 (50 μM) for 6 hours. Cell lysates were subjected to immunoblot with Myc or Flag antibody. (L) HEK293T cells transfected with various combinations of plasmids as indicated below each column were left untreated or treated with MG132 for 6 hours. The relative luciferase activities were measured after 48 hours by a dual-luciferase reporter assay. All activities were normalized to pGL3-Basic activity. (M) HEK293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-EKLF, Flag-GPS2, and hemagglutinin-ubiquitin (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. (N) MEL cells with or without stable GPS2 knockdown were pretreated with MG132 (20 μM) for 6 hours before collection. The cell lysates were immunoprecipitated with tandem ubiquitin binding entities (TUBEs)-conjugated agarose beads and then subjected to immunoblot analysis of EKLF ubiquitination. Data are representative of 3 independent experiments (A-B,K,M-N). All values are mean ± SEM (n = 3 replicates). *P < .05, **P < .01, ***P < .001; 2-tailed unpaired t test (C-D,L) or 2-way ANOVA test (E-J).

GPS2 increases protein stability of EKLF but does not alter its ubiquitination. (A-B) Immunoblot analysis of GPS2 and EKLF protein levels in GPS2-knockdown and control MEL cells (A), as well as in Gps2+/+ and Gps2−/− E12.5 fetal livers (B). (C-D) Real-time PCR analysis of GPS2 and EKLF mRNA levels in GPS2-knockdown and control MEL cells (C), as well as in Gps2+/+ and Gps2−/− E12.5 fetal livers (D). (E) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2 or control vector were treated with CHX for indicated times and then analyzed by western blot. (F-G) MEL cells with/without stable overexpression of GPS2 (F) or knockdown of GPS2 (G) were treated with CHX for indicated times and then analyzed by western blot. (H) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2-Δ111-150 or control vector were treated with CHX for indicated times and then analyzed by western blot. (I) HEK293T cells transfected with Myc-EKLF-Δ191-230 together with Flag-GPS2 or control vector were treated with CHX for indicated times and then analyzed by immunoblot. (J) HEK293T cells transfected with Myc-EKLF-Δ1-68 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. (K) HEK293T cells transfected with Myc-EKLF together with Flag-GPS2 or control vector were treated with vehicle, MG132 (20 μM), or E64 (50 μM) for 6 hours. Cell lysates were subjected to immunoblot with Myc or Flag antibody. (L) HEK293T cells transfected with various combinations of plasmids as indicated below each column were left untreated or treated with MG132 for 6 hours. The relative luciferase activities were measured after 48 hours by a dual-luciferase reporter assay. All activities were normalized to pGL3-Basic activity. (M) HEK293T cells were transfected with various combinations (above lanes) of plasmids encoding Myc-EKLF, Flag-GPS2, and hemagglutinin-ubiquitin (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. (N) MEL cells with or without stable GPS2 knockdown were pretreated with MG132 (20 μM) for 6 hours before collection. The cell lysates were immunoprecipitated with tandem ubiquitin binding entities (TUBEs)-conjugated agarose beads and then subjected to immunoblot analysis of EKLF ubiquitination. Data are representative of 3 independent experiments (A-B,K,M-N). All values are mean ± SEM (n = 3 replicates). *P < .05, **P < .01, ***P < .001; 2-tailed unpaired t test (C-D,L) or 2-way ANOVA test (E-J).

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