f9 mouse embryonal carcinoma cell line  (ATCC)

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SCF FBXL14 complex stimulates neuronal differentiation by targeting the Notch signaling factor HES1 for proteolysis

doi: 10.1074/jbc.M117.815001

Figure Lengend Snippet: The WRPW motif was required for HES1 ubiquitination and degradation. A, highly conserved WRPW motif and schematic diagram for HES1 ΔC mutant. Six amino acids were deleted on the C-terminal of HES1. B, deletion of WRPW motif reduced the interaction between HES1 and FBXL14. 293T cells were co-transfected with pEGFPC2–FBXL14 and pMSCV–3× FLAG–3× HA–HES1 or pMSCV–3× FLAG–3× HA–HES1 ΔC. Co-immunoprecipitation (IP) was performed using anti-GFP and anti-FLAG antibodies. The experiment was biologically repeated at least three times. C, predicted binding mode of FBXL14 and the WRPW motif. Peptide-binding interface of the FBXL14 leucine-rich domain, from the MD simulation. The CH3 group of blocked acetyl at N terminus is shown as a sphere. Carboxyl oxygen and amino nitrogen atoms are colored red and blue, respectively. D, detailed view of the key interactions between the WRPW motif and FBXL14 leucine-rich domain. E, the WRPW motif was critical for HES1 degradation. Stably expressing 3× FLAG–3× HA–HES1 WT or 3× FLAG–3× HA–HES1 ΔC F9 cells were treated with CHX for the indicated times, and then the cells were harvested for Western blotting to detect the protein level changes of HES1. The error bars represent S.D. from three independent experiments. F, SCFFBXL14 complex did not degrade HES1 ΔC mutant. Stably expressing 3× FLAG–3× HA–HES1 or 3× FLAG–3× HA–HES1 ΔC mutant F9 cells were transfected with luciferase, RBX1, CUL1, or FBXL14 siRNAs. Cell lysates were immunoblotted (IB) for the indicated proteins. The protein levels in the blots were quantified and plotted at the bottom. The error bars represent S.D. from three independent experiments. The significance of statistical difference was calculated by paired two-sided Student's t test (**, p < 0.01). G, WRPW motif was essential for SCFFBXL14 catalyzing HES1 ubiquitination. 293T cells were co-transfected with pMSCV–3× FLAG–3× HA–HES1 or pMSCV–3× FLAG–3× HA–HES1 ΔC, pEGFPC2–FBXL14, or pEGFPC2–FBXL14 ΔF and pRK5-HA-Ub as indicated for 44 h followed by incubated with MG132 for 4 h, and the cells were harvested for immunoprecipitation using anti-HES1 antibody under denaturing condition and immunoblotted with anti-HA antibody. Whole-cell extracts (WCE) were immunoblotted with anti-GFP and anti-HA antibodies to indicate expression of exogenous FBXL14 and HES1. The experiment was biologically repeated at least three times.

Article Snippet: Mouse embryonic stem cell RW.4, mouse embryonal carcinoma F9 cells, human kidney 293T cells were purchased from American Type Culture Collection and cultured as described ( 51 ).

Techniques: Ubiquitin Proteomics, Mutagenesis, Transfection, Immunoprecipitation, Binding Assay, Stable Transfection, Expressing, Western Blot, Luciferase, Incubation

Journal: The Journal of Biological Chemistry

Article Title: The E3 ubiquitin ligase SCF FBXL14 complex stimulates neuronal differentiation by targeting the Notch signaling factor HES1 for proteolysis

doi: 10.1074/jbc.M117.815001

Figure Lengend Snippet: Lysines 83 and 106 were potential ubiquitination sites for SCFFBXL14-mediated HES1 ubiquitination. A, lysine mutants screening. Stably expressing 3× FLAG–3× HA–HES1 WT or mutants (as indicated) F9 cells were transfected with luciferase or CUL1 siRNAs for 45 h, and cell lysates were immunoblotted using indicated antibodies. Histone H3 was taken as loading control. B, quantification of the relative protein levels of HES1 and mutants in A. The quantification of protein levels was measured as in Fig. 1A. The statistical difference between the control group (WT) and experimental groups (K24A, K64A, K71A, K83A, K106A, and K83A/K106A) was measured. The error bars represent S.D. from three independent experiments. The significance of statistical difference was calculated by paired two-sided Student's t test (**, p < 0.01). C, HES1 was ubiquitinated by FBXL14 through lysines 83 and 106. 293T cells were co-transfected with pMSCV–3× FLAG–3× HA–HES1 or pMSCV–3× FLAG–3× HA-0HES1 (K83A/K106A), pEGFPC2–FBXL14, and pRK5-HA-Ub as indicated for 44 h followed by incubation with MG132 for 4 h, and the cells were harvested for immunoprecipitation (IP) using anti-HES1 antibody under denaturing condition and immunoblotted with anti-HA antibody. Whole-cell extracts (WCE) were immunoblotted with anti-GFP and anti-HA antibodies to indicate expression of exogenous FBXL14 and HES1. The experiment was biologically repeated at least three times.

Article Snippet: Mouse embryonic stem cell RW.4, mouse embryonal carcinoma F9 cells, human kidney 293T cells were purchased from American Type Culture Collection and cultured as described ( 51 ).

Techniques: Ubiquitin Proteomics, Stable Transfection, Expressing, Transfection, Luciferase, Control, Incubation, Immunoprecipitation