Fig 1: CUL4ADET1-COP1 interacts with Sox2 and regulates its stability. a Network view of E3–Sox2 interactions (left panel) and the E3 hierarchical tree for Sox2 (right panel). UbiBrowser was employed to explore the E3 ligases for Sox2. The representative predicted E3 ligases surround Sox2. The node colors and characters reflect the E3 type. The edge width, the node size, and the edge shade are corrected with the confidence score. The predicted E3s and their position in the E3 family hierarchical tree was presented. In this tree, texts in each circle (just like “U”, “D” and “SO”) represent the E3 family. The number in the bracket following each E3 family represents the number of corresponding predicted E3–Sox2 interaction. b NPCs cell lysates were subjected to immunoprecipitation with control IgG or anti-Sox2 antibodies and detected CUL4A, COP1, DET1, DDB1, Roc1, and Sox2 protein levels. c The lysates of HEK293T cells transfected with indicated constructs were subjected to immunoprecipitation with anti-Myc or Histidine tag-specific affinity resin (agarose beads). The immunoprecipitates or the eluates were then blotted. d Overview of the structures of COP1 wild type and different truncates. HEK293T cells were co-transfected with Myc-Sox2 and the indicated COP1 truncates. The lysates were collected and subjected to immunoprecipitation with anti-Flag. The immunoprecipitates were then blotted. e Overview of the structure of Sox2 wild type and different VP mutants. Recombinant proteins (His-COP1, GST-Sox2, GST-Sox2-A1, GST-Sox2-A2, and GST-Sox2-AA) were expressed and purified. GST-Sox2 bound to glutathione-Sepharose 4B beads was incubated with His- COP1 for 24 h at 4 °C. Then the beads were washed and proteins were eluted, followed by western blotting. f HEK293T cells were transfected with indicated constructs. The lysates were collected and blotted with anti-Flag and anti-Myc antibody. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Fig 2: COP1 interacts with Sox2 directly. a Cells were transfected with indicated constructs and siRNA. Flag-Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. b HEK293T cells were transfected with indicated constructs and siRNA, and Sox2 was immunoprecipated with anti-Flag and immunoblotted with anti-Myc. c HEK293T cells were transfected with indicated constructs and western blot was performed to measure the expression of Flag-Sox2, Flag-COP1, and Myc-DET1. d Increasing amounts of Flag-COP1 were co-transfected together with Flag-Sox2 and DET1 into HEK293T cells and ectopic Flag-Sox2 expression was detected. e The predicted work model of CUL4ADET1-COP1 for Sox2 degradation. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Fig 3: CUL4ADET1-COP1 ubiquitylates SOX2. a HEK293T cell line with knockdown of COP1 (upper panel) or DET1 (lower panel) were treated with CHX (10 µg/ml), and collected at the indicated times for western blot. Quantification of Sox2 level relative to tubulin is shown. Results are shown as mean ± s.d. n = 3 independent experiments. **P < 0.01, two-way ANOVA test. b HA-Ub was co-transfected with indicated siRNA into HEK293T cells. Cells were treated with MG132 for 8 h before collection. Sox2 was immunoprecipitated with anti-Sox2 and the ubiquitylated Sox2 proteins were immunoblotted with anti-HA. c Cell-free Sox2 ubiquitylation assay. The purified GST-Sox2, CUL4A, COP1, and DET1 proteins were incubated with commercial E1, UbE2D3 (E2), and ubiquitin for 2 h at 37 °C. The mixtures were subjected to GST pull-down and western blot with anti-His antibody. d Immunoblotting of Flag-Sox2 in cells transfected with indicated constructs. Quantification of Sox2 level is shown under the blot. e Cells were transfected with indicated siRNA and the cell lysates were subjected to western blot to detect Sox2 expression. Quantification of relative protein level is shown under the blot. The representative images are shown from three independent experiments. Unprocessed original scans of blots are shown in Supplementary Fig. 9
Supplier Page from ABclonal Technology for DET1 Rabbit pAb
Trial Size: 20 ul