Journal: Molecular Biology of the Cell
Article Title: ZFP36L1 Negatively Regulates Erythroid Differentiation of CD34+ Hematopoietic Stem Cells by Interfering with the Stat5b Pathway
doi: 10.1091/mbc.E10-01-0040
Figure Lengend Snippet: ZFP36L1 binding to AU-rich-elements (ARE) in the 3′UTR of Stat5b mRNA confers instability to mRNAs containing such elements. (A) Left, Western blot demonstrating identity and integrity of in vitro–translated FlagZFP36L1 protein: immunoblotting was performed with anti-Flag or anti-ZFP36L1 antibody as indicated. Right, RNA mobility shift assay performed by incubating in vitro–translated FlagZFP36L1 protein with labeled RNA probes corresponding to ARE in the 3′UTR of GM-CSF mRNA (used as a positive control, lanes 1–4) or to ARE in the 3′UTR of Stat5b mRNA (lanes 5–8). Probe sequences are: GM-CSF: 5′-UAUUUAUUUAUUUAUUUAUUUA-3′; Stat5b: 5′-AUAGUAAAUUAUUUAUUGGAAGAU-3′. Supershifts were obtained by an additional incubation with anti-Flag Ab (lanes 2 and 6). Competition experiments were performed with cold Stat5b probe (lane 3) or with cold GM-CSF probe (lane 7). Lanes 4 and 8 represent labeled probes incubated with an in vitro translation reaction mix performed on empty vector. (B) Luciferase activity assay performed in HEK293 cells transfected with pcDNA3.1 empty expression vector or with pcDNA3.1 overexpressing FlagZFP36L1 (20 ng) together with pGL3 reporter construct encoding for a luciferase gene fused to the 3′UTR of Vegfa (positive control) or to the 3′UTR of Stat5b. Luciferase activity is represented in terms of fold change; error bars, SEM calculated on a set of five independent experiments. (C) Ribonucleoprotein complexes immunoprecipitation assay demonstrating that binding of ZFP36L1 protein to the ARE in the 3′UTR of Stat5b mRNA occurs in vivo: ribonucleoprotein complexes were immunoprecipitated from lysates of HEK293 cells transfected with pcDNA3.1 ZFP36L1 and pGL3 3′UTR Stat5b vectors, RNA was extracted, reverse-transcribed, and amplified by PCR using primers specific for Stat5b 3′UTR fused to the luciferase gene. Lanes 1 and 2, negative and positive control respectively, i.e., PCR amplification performed on no template or on RNA extracted from cell lysates before immunoprecipitation; lane 3, specific immunoprecipitation obtained with anti-ZFP36L1 antibody; lanes 4 and 5, control immunoprecipitations performed with nonspecific antibody or with no antibody.
Article Snippet: Supershifts were determined by an extra 20-minute incubation in the presence of 1 μg Flag M2 mouse MoAb (Sigma-Aldrich).
Techniques: Binding Assay, Western Blot, In Vitro, Mobility Shift, Labeling, Positive Control, Incubation, Plasmid Preparation, Luciferase, Activity Assay, Transfection, Expressing, Construct, Immunoprecipitation, In Vivo, Amplification
