flirt  (MathWorks Inc)

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: ( a ) Structure of AF10, AF10/CALM, CALM/AF10 and CALM. BP: breakpoint; PHD: plant homeo domain; ePHD: extended plant homeo domain; OM/LZ: octapeptide/leucine zipper; ANTH: AP180 N-terminal homology domain; CID: CATS interaction domain. The BP in AF10 and CALM is indicated by arrows. ( b ) Structure of the FHL2 protein. FHL2 is composed of four and a half LIM domains.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques:

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: ( a ) GST-pulldown experiment: bead-bound GST-FHL2 was incubated with 35 S-labeled CALM (1–408), and the beads were washed and analyzed by SDS-PAGE. Only GST-FLH2 (lane 3), but not GST alone (lane 2), is able to retain 35 S-labeled-CALM (1–408). ( b ) Co-immunoprecipitation (co-IP) of FLAG-FHL2 and YFP-CALM. HEK293T cells were transiently co-transfected with plasmids expressing FLAG-FHL2 and YFP-CALM or FLAG-FHL2 and YFP. In lanes 1 and 3, 10% of the protein extract that was used for each co-IP reaction is loaded. GFP-binder beads were used for the precipitation, and only YFP-CALM (lane 2), but not YFP alone (lane 4), is able to precipitate FLAG-FHL2.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Incubation, Labeling, SDS Page, Immunoprecipitation, Co-Immunoprecipitation Assay, Transfection, Expressing

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: ( a ) FHL2 prey clone: alignment of the FHL2 prey clone with FHL2 (GAL4AD-FHL2). UTR: untranslated region; ORF: open reading frame; AD: activation domain; nt: nucleotide. Numbers above the diagrams refer to amino acids, and that below refer to nucleotides. ( b ) Mapping of the FHL2 interaction domain of CALM. A series of CALM deletion mutants was expressed as bait proteins in yeast together with the FHL2 prey protein (GAL4AD-FHL2). Dilutions of the transformants were spotted on yeast synthetic drop-out medium lacking tryptophan and leucine (–W/–L) to control for double transformant status or lacking tryptophan, leucine, histidine and adenine (–H/–A/–L/–W) to assay the activation of the reporter genes. Only the yeast clones with the CALM mutants CALM (1–408) and CALM (1–335) grow on the –H/–A/–L/–W plates, indicating that amino acids 294–335 of CALM are necessary for its interaction with FHL2. ( c ) Mapping of the CALM interaction domain of FHL2. Structure of CALM prey clone obtained from the yeast two-hybrid assay, GAL4 AD-CALM. AD: activation domain. A series of FHL2 deletion mutants was expressed as bait proteins in yeast together with the CALM prey protein (GAL4AD-CALM). Yeast double transformants were streaked on yeast synthetic drop-out medium lacking tryptophan and leucine (–W/–L) to control for double transformant status or on SD plates lacking tryptophan, leucine, histidine and adenine (–H/–A/–L/–W) to assay the activation of the reporter genes. Only the yeast clones with the full-length FHL2 (clone (1)) grow on the –H/–A/–L/–W plates, indicating that amino acids 206–279 of FHL2 are required for interaction with CALM.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Activation Assay, Clone Assay, Y2H Assay

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: Subcellular localization of FHL2, CALM and CALM/AF10. Confocal images of U2OS cells transiently transfected with plasmids expressing the proteins indicated on the left-hand side. Channels are indicated above the images (4′,6-diamidino-2-phenylindole, YFP, Cy3 and merged). For a detailed explanation of this figure, see the Results section.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Transfection, Expressing

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: Line scans of the confocal images m, q and u from to demonstrate colocalization of ( a ) FLAG-FHL2 with YFP-CALM; ( b ) FLAG-FHL2 with YFP-CALM/AF10; and ( c ) YFP-FHL2 with FLAG-CALM/AF10.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques:

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: ( a ) CALM inhibits FHL2-dependent activation of transcription. HEK293T cells were transiently transfected with plasmids expressing GAL4DBD, GAL4DBD-FHL2 and YFP-CALM, a luciferase reporter plasmid and a Renilla luciferase control plasmid as indicated. The bars indicate fold activation of luciferase activity compared to GALDBD. Four independent experiments with three measurements per sample were performed. The error bars represent standard deviations. The firefly luciferase values were normalized to the Renilla luciferase values. ( b ) CALM/AF10 does not inhibit FHL2-dependent activation of transcription. HEK293T cells were transiently transfected with plasmids expressing GAL4DBD, GAL4DBD-FHL2 and YFP-CALM/CAF10 and a luciferase reporter plasmid as indicated. Co-transfection of YFP-CALM/AF10 did not have a significant influence on the transcriptional activation mediated by GAL4DBD-FHL2. ( c ) FHL2 inhibits CALM/AF10-dependent activation of transcription. HEK293T cells were transiently transfected with plasmids expressing GAL4DBD, GAL4DBD-CALM/AF10, YFP-CALM/AF10 and a luciferase reporter plasmid as indicated. In this setting, GAL4DBD-CALM/AF10 is a strong transcriptional activator. A threefold reduction of the GAL4DBD-CALM/AF10-mediated transcriptional activation is observed when FLAG-FHL2 is co-expressed. Three independent experiments were performed and measured in duplicate.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Activation Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Activity Assay, Cotransfection

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: Expression of FHL2 in leukemia patient samples. FHL2 is expressed at higher levels in CML and AML with complex aberrant karyotype. A microarray analysis was performed on 129 patient samples and 10 normal bone marrow (nBM) samples. The boxplots depict the expression levels of FHL2 in nBM, CML, CALM/AF10-positive leukemias, in seven different AML subtypes (AML_nk: AML with normal karyotype; AML_comp: AML with complex aberrant karyotype; AML_MLL: AML with an MLL rearrangement; AML_M4: AML with CBFB/MYH11 fusion; AML_M3: acute promyelocytic leukemia with a PML/RARA fusion; AML_M2: AML with an AML1/ETO fusion; AML_FLT3: AML with normal karyotype and with an fms-like tyrosine kinase receptor-3 (FLT3) internal tandem duplication), and four ALL subtypes (ALL_Ph: ALL with BCR/ABL fusion; ALL-MLL: ALL with an MLL rearrangement; ALL_BA: c-ALL: (common ALL) and ALL: pro-B-ALL). Each group is composed of 10 patient samples, except the ALL-MLL patient group, which contains 9 samples. Expression intensities are depicted on a logarithmic scale. The median of the distribution is indicated by a bar. The box presents 50% of the expression values in that group and is called the interquartile range (IQR). An expression value is considered an outlier when it lies more than 1.5 IQR lower or higher than the first or third quartile, respectively. Outliers are represented as circles. The smallest and largest values that are not outliers are indicated by a vertical tic mark or ‘whisker', which is connected to the box via a horizontal line. If the notches of the boxes do not overlap between two groups, there is evidence that the medians are significantly different between the groups.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Expressing, Microarray, Whisker Assay

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: Expression of FHL2 in samples from patients with AML with complex aberrant karyotypes, AML M6 and AML with normal karyotypes. FHL2 microarray expression levels from 308 AML. Group 1 consists of 167 AML patients with normal karyotypes of all FAB subtypes, except M6, group 2 consists of 115 samples from patients with AML with complex aberrant karyotypes, and group 3 contains 26 samples from patients with AML M6. FHL2 is significantly higher expressed in patients with AML M6 than in AML patients with a normal karyotype or in patients with complex aberrant karyotypes ( P =2.2 × 10 −16 and P =9.967 × 10 −10 , respectively). A significant difference in the expression of FHL2 was found between AML patients with normal karyotype and patients with AML complex karyotype with a P -value=1.04 × 10 −12 .

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Expressing, Microarray

Journal: Blood Cancer Journal

Article Title: FHL2 interacts with CALM and is highly expressed in acute erythroid leukemia

doi: 10.1038/bcj.2011.40

Figure Lengend Snippet: FHL2 is highly expressed in patients with AML M6 karyotype. FHL2 expression was determined using qRT-PCR in 45 AML patient samples. High expression of FHL2 was observed in patients with AML M6 in comparison to patients with complex or normal karyotype ( P -value: 0.000). No statistical significance was discovered between AML patients with complex karyotype and patients with normal karyotype ( P -value: 0.685). The data are presented as fold change in gene expression compared to the gene expression of AML patients with normal karyotype.

Article Snippet: The GST-Flirt 1 (full-length FHL2) was expressed in the Escherichia coli strain XL1 blue (Agilent Technologies, Böblingen, Germany) and purified from bacterial proteins by incubation with glutathione Sepharose 4B beads (GE Healthcare Bio-Sciences, Munich, Germany).

Techniques: Expressing, Quantitative RT-PCR