Research Paper Volume 13, Issue 10 pp 13739—13763

Glycogen synthase kinase-3 beta (GSK3β)-mediated phosphorylation of ETS1 promotes progression of ovarian carcinoma

The GSK3β phosphorylation complex includes ETS1. (A) The endogenous GSK3β-ETS1 complex was pulled-down in ovarian cancer cell lysates (MDAH2774) using an anti-ETS1 antibody (left panel), an anti-GSK3β antibody (right panel), and a control IgG. The associated GSK3β protein (ETS1 antibody pull-down, left panel) or ETS1 (GSK3β antibody pull-down, right panel) were determined by western blot. Levels of endogenous GSK3β and ETS1 are shown in the lower panel. (B) Confocal microscopy revealed the presence of a GSK3β-ETS1 complex in MDAH2774 cells. Cells were double-stained with the GSK3β antibody (green signal, left panel), the ETS1 antibody (red signal, middle lower panel), or the control IgG (middle upper panel). Two proteins showed colocalization (yellow signal) as revealed by overlapping green and red signals (right panel). (C) 293 cells were transfected with full-length ETS1 (Halo-ETS1) and control vector, the full length and truncated GSK3β constructs (CBP tag). The presence of ETS1 aggregated in a complex was examined with an anti-Halo tag antibody. Asterisks indicate the full length and truncated GSK3β protein. The reference number of amino acid sequence for GSK3β isoform 1 is NP

Figure 1. The GSK3β phosphorylation complex includes ETS1. (A) The endogenous GSK3β-ETS1 complex was pulled-down in ovarian cancer cell lysates (MDAH2774) using an anti-ETS1 antibody (left panel), an anti-GSK3β antibody (right panel), and a control IgG. The associated GSK3β protein (ETS1 antibody pull-down, left panel) or ETS1 (GSK3β antibody pull-down, right panel) were determined by western blot. Levels of endogenous GSK3β and ETS1 are shown in the lower panel. (B) Confocal microscopy revealed the presence of a GSK3β-ETS1 complex in MDAH2774 cells. Cells were double-stained with the GSK3β antibody (green signal, left panel), the ETS1 antibody (red signal, middle lower panel), or the control IgG (middle upper panel). Two proteins showed colocalization (yellow signal) as revealed by overlapping green and red signals (right panel). (C) 293 cells were transfected with full-length ETS1 (Halo-ETS1) and control vector, the full length and truncated GSK3β constructs (CBP tag). The presence of ETS1 aggregated in a complex was examined with an anti-Halo tag antibody. Asterisks indicate the full length and truncated GSK3β protein. The reference number of amino acid sequence for GSK3β isoform 1 is NP_002084.2 while the nucleotide number is NM_002093.4. (D) Full-length GSK3β (HA-GSK3β) or control vector was co-transfected with the full length and truncated ETS1 constructs (FL: full length, 1−378, 1−197, 191−486, and 363−486) into 293 cells. Upon purification with streptavidin beads, co-precipitated GSK3β was detected using an anti-HA antibody. The reference number of amino acid sequence for ETS1 is NP_001137292.1.