tf1 acc 334 cell lines  (DSMZ)

Journal: Nature genetics

Article Title: Recurrent SETBP1 mutations in atypical chronic myeloid leukemia

doi: 10.1038/ng.2495

Figure Lengend Snippet: Interaction between β-TrCP1 and SETBP1. (a) The β-TrCP1 degron motif (amino acids 868–873) is highlighted in red on the SETBP1 protein schematic. The sequences of biotinylated phosphorylated peptides (amino acids 859–879) used in the experiments are given. Black circles represent alterations found in aCML samples; green circles represent alterations found in other diseases. (b) Peptide pulldown experiment performed using TF1 total cell lysate and phosphorylated peptides. Beads with no peptides were used to control for nonspecific binding. Immunoblotting for β-TrCP1 was performed on the bound fractions. Immunoblotting for actin on the unbound fractions was used as a loading control. (c) Peptide pulldown experiment using recombinant SCF–β-TrCP1 complex on phosphorylated (+ P) and dephosphorylated (− P) peptides representing either wild-type SETBP1 or SETBP1 Gly870Ser. Beads with no peptide were used to control for nonspecific binding. Immunoblotting for β-TrCP1 was performed on bound as well as unbound (control) fractions.

Article Snippet: The TF1 human erythroleukemia cell line was purchased from DSMZ and maintained in RPMI 1640 medium (Lonza Cambrex) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 80 μg/ml gentamicin, 20 mM HEPES and 2 ng/ml human GM-CSF (Life Technology).

Techniques: Control, Binding Assay, Western Blot, Recombinant

Journal: Nature genetics

Article Title: Recurrent SETBP1 mutations in atypical chronic myeloid leukemia

doi: 10.1038/ng.2495

Figure Lengend Snippet: Effects of the SETBP1 Gly870Ser alteration on SETBP1 and SET protein expression, PP2A activity and cell growth. (a–d) TF1 cells were transfected with empty vector (pMIGR1, EV) or vector expressing wild-type SETBP1 or SETBP1 Gly870Ser. (a) Immunoblotting for SETBP1 on whole-cell lysates. Whole normal fetal stomach lysate was used as a control for SETBP1 protein. Immunoblotting for actin was used as a loading control. Lanes were derived from the same gel and were juxtaposed. (b) Immunoblotting for SET, phosphorylated PP2A (pPP2A), PP2A and actin on whole-cell lysates. Immunoblotting for actin was used as a loading control. Densitometric analysis of the amount of SET protein normalized over actin signal is shown in the bar graph. Mean and s.e.m. values from three independent experiments are plotted. ***P < 0.0001 compared to cells expressing wild-type SETBP1. (c) Lysates were used to assess the activity of PP2A. The activity relative to cells expressing wild-type SETBP1 is reported. Mean and s.e.m. values from three independent experiments are plotted. ***P < 0.0001. (d) Growth rate of the cells as measured by tritiated thymidine incorporation. Each curve was normalized to its value at time 0. Mean and s.e.m. values from two independent experiments are plotted. ***P < 0.0001 compared to cells expressing wild-type SETBP1.

Article Snippet: The TF1 human erythroleukemia cell line was purchased from DSMZ and maintained in RPMI 1640 medium (Lonza Cambrex) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin G, 80 μg/ml gentamicin, 20 mM HEPES and 2 ng/ml human GM-CSF (Life Technology).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Control, Derivative Assay