rabbit polyclonal anti eya2  (Abcam)

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Myeloid immortalization of KSL and MP cells by Plzf in association with high Eya2 expression. (A) Experimental strategy for gene expression profiling of Plzf-transduced cells. (Middle panel) Lin-depleted (Lin−) BM cells displayed with the sorting gate for KSL and MP cells. Both subpopulations were retrovirally transduced with Plzf-IRES-EGFP (Plzf-IG) or EGFP (empty [IG]), in pMYs-IG on day 0 (d0). (Left [MP] and right [KSL] panels) Green fluorescent protein (GFP)-positive cells (blank area for Plzf-IG, gray shading for IG) were sorted on day 3 (d3). (B) Expression levels of Eya family genes in Plzf-transduced cells by RT-qPCR normalized to Actb. *, P < 0.05; **, P < 0.005; n.s., not significant. (C) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of Plzf or a Plzf mutant lacking its BTB/POZ domain (PlzfΔBTB). pMYs-IRES-Neomycinr (pMYs-IN) and pMYs-IRES-puromycinr (pMYs-IP) were used as backbone vectors (empty) in panels D and F and panel E, respectively. (D and E) Expression levels of Eya2 by RT-qPCR normalized to Actb in the myeloid immortalization assays of Plzf-transduced cells (D) and Plzf- or PlzfΔBTB-transduced KSL cells (E), respectively. (F) Expression levels of Eya2 protein (upper) in comparison with those of Eya2 transcripts (lower). In two independent experiments (Exp. 1 and 2) with the immortalization assays, cell lysates and RNA prepared from colony-forming cells were subjected to Western blotting using anti-Eya2 and anti-Stat5a (an internal control) antibodies (αEya2 and αStat5a) and RT-qPCR analyses, respectively. Eya2-immortalized KSL cells (E2-IC [described in detail later]) were used as a positive control. Representative blots (Exp. 1) are shown in the upper panels. Bar graphs show means ± standard deviations (SD) from three independent experiments in panels B, D, and E.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Transduction, Quantitative RT-PCR, Mutagenesis, Western Blot, Positive Control

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Localization of Plzf in the putative promoter region of Eya2. (A) Overview of the genomic region covering Eya2 and its upstream sequence. In an adapted UCSC Genome Browser view (chromosome 2: 165,333,718 to 165,601,417 [on mm9]), three kinds of Eya2 transcripts are shown in the RefSeq gene track (top). ChIP-seq data from mouse thymus (middle) and bone marrow (BM) (bottom) are shown in the corresponding LICR TFBS (Pol II and input control) and histone (H3K4me3, H3K4me1, H3K27ac, and input control) tracks. (B) Genomic structure of alternative first exons of Eya2. Primer sets (for the regions of a, b, c-1, c-2, c-3, and c-4 in ChIP-qPCR shown in panel D) were designed around these exons. Arrowheads indicate primers detecting alternative transcripts of Eya2 in panel C. (C) Detection of alternative transcripts of Eya2 by RT-PCR in Plzf-immortalized KSL cells. NTC, negative control. (D) Relative binding of Plzf (detected by anti-FLAG antibody) and RNA polymerase II around exon 1c of Eya2 in KSL (left) and MP (right) cells immortalized by FLAG-tagged Plzf. The promoter region of Hbb-b1 was examined as a negative control. *, P < 0.05. Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Sequencing, ChIP-sequencing, Reverse Transcription Polymerase Chain Reaction, Negative Control, Binding Assay

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Myeloid immortalization of myeloid progenitor cells by PLZF-RARA in association with Eya2 expression. (A) Experimental strategy for myeloid immortalization assays of Sca-1/Lin-depleted myeloid progenitor cells. The cells were retrovirally transduced with PLZF, PLZF-RARA, PML-RARA, and Hoxa9-IRES-Mesi1 (Hoxa9/Meis1) in pMYs-IN. (B) Expression of PLZF, PLZF-RARA, and PML-RARA by Western blotting analyses. Lysates extracted from Plat E cells transfected with empty vector (pMYs-IN), pMYs-PLZF-IN, or pMYs-PLZF-RARA-IN were blotted with anti-PLZF antibody (αPLZF), followed by being reprobed with anti-α-tubulin antibody (αTub) as an internal control (upper panels). Lysates extracted from Plat E cells transfected with empty (pMYs-IN) or pMYs-PML-RARA-IN were immunoprecipitated with anti-RARA antibody (αRARA), followed by blotting with anti-RARA (lower panel). (C and D) Expression levels of Eya2 (C) and Bcl2, c-Myc, and Cebpε (D) by RT-qPCR in the myeloid immortalization assays. (E) Relative expression levels of Eya2 by RT-qPCR in the retrovirally transduced cells at the end of the first plating. Normalization was performed using primers for a part of the PLZF portion of PLZF-RARA. (F) Degradation of PLZF-RARA by ATRA in PLZF-RARA-immortalized cells by Western blotting analyses. Lysates extracted from these cells with 1 μM ATRA or vehicle control (EtOH) for 16 h were blotted with anti-PLZF, followed by being reprobed with anti-Stat5a as an internal control. (G) Time course of cell proliferation (left) and expression levels of Eya2 by RT-qPCR (right) in PLZF-RARA-immortalized cells with 1 μM ATRA or vehicle control (EtOH). (H) Effect of ATRA on clonogenicities (3,000 cells/dish [left]) and expression levels of Eya2 by RT-qPCR (right) in PLZF-RARA-immortalized cells. Bar graphs show means ± SD from three independent experiments. *, P < 0.05; **, P < 0.005; n.s., not significant.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Transduction, Western Blot, Transfection, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Myeloid immortalization of KSL and MP cells by Eya2. (A) Experimental strategy for myeloid immortalization assays with retroviral transduction using pMYs-IN. (B) Structure of Eya2 and missense mutants. Arrows show primers used in RT-qPCR. (C) Expression of Eya2 and its mutants by Western blotting analyses using anti-Eya2 and anti-α-tubulin (αTub [an internal control]) antibodies (αEya2 and αTub). (D) Expression levels of Eya2 and its mutants by RT-qPCR of colony-forming cells at the end of the first plating in panel A. (E) Myeloid immortalization assays of KSL and MP cells after retroviral transduction. (F to H) Typical morphology of colonies of Eya2-transduced KSL cells at the third round of plating (F) and typical morphology (G) and immunophenotype (H) of the cells constituting the colonies. Cells were stained with Wright-Giemsa stain. (I) Localization of Eya2 in the FLAG-tagged Eya2-immortalized KSL cells analyzed by immunofluorescent confocal microscopy. Alexa Fluor 568-conjugated secondary antibody reacting with anti-DDDDK-tag antibody (αDDDDK) in the immortalized cells visualized their cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and a merged image is displayed (bottom). Magnifications (bar lengths): F, ×40 (200 μm); G, ×400 (20 μm); I, ×400 (30 μm). Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Staining, Giemsa Stain, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Reduced clonogenicity of PLZF-RARA-immortalized cells with Eya2 depletion. (A) Experimental strategy for analysis of PLZF-RARA-immortalized cells with Eya2 depletion by retroviral transduction of shRNA/Kusabira-Orange gene (KO) coexpressor in pMXsU6-KO. (B and C) Expression levels of Eya2 by RT-qPCR (B) and relative CFU (3,000 cells/dish) (C) of the cells sorted from shRNA-transduced cells on day 2. (D and E) Expression levels of Eya2, Cebpε, Bcl2, and c-Myc by RT-qPCR (D) and immunophenotypes and apoptotic subpopulations by FACS analyses (E) of shRNA-transduced cells expressing KO on day 4. Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Transduction, shRNA, Expressing, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Involvement of Six1 in myeloid immortalization by Eya2. (A) Experimental strategy for analysis of Eya2-immortalized KSL cells with retroviral transduction of Six1 or the Six1 mutant lacking its homeodomain (Six1ΔHD) using pMYs-IP. puro, puromycin. (B) Structure of Six1 and Six1ΔHD and their expression by Western blotting analyses using anti-Six1 antibody (αSix1) and anti-α-tubulin (αTub [an internal control]). Horizontal lines indicate the regions amplified by RT-qPCR. SD, Six domain; HD, homeodomain. (C and D) Expression levels of Six1 and Six1ΔHD by RT-qPCR (C) and relative CFU (D [3,000 cells/dish]) of the Eya2-immortalized cells forced to express Six1 or Six1ΔHD.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Transduction, Mutagenesis, Expressing, Western Blot, Amplification, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Inducible immortalization of KSL and MP cells by ER-Eya2. (A) Experimental strategy for myeloid immortalization assays of KSL and MP cells with retroviral transduction of the ER-Eya2 fusion gene using pMYs-IN. ER, estrogen receptor gene; 4OHT, 4-hydroxytamoxifen. (B) Structure of Eya2 and ER-Eya2. LBD, mutant ligand-binding domain of the mouse ER. (C) Expression of ER-Eya2 by Western blotting using anti-Eya2 (αEya2 [top]), anti-ER antibody (αER [middle]), and anti-α-tubulin (αTub [an internal control; bottom]). (D) Myeloid immortalization assays of ER-Eya2-transduced cells in the presence or absence of 4OHT. (E) 4OHT-dependent clonogenicity of cells inducibly immortalized by ER-Eya2. (F) Localization of ER-Eya2 in the inducibly immortalized KSL cells analyzed by immunofluorescent confocal microscopy, following a 3-day culture in the presence (left panels) and absence (right panels) of 4OHT. Alexa Fluor 568-conjugated secondary antibody reacting with anti-ER in the immortalized cells visualized its cellular localization (top). Nuclei were visualized with TO-PRO-3 iodide (middle), and merged images are displayed (bottom). Magnification (bar length), ×400 (30 μm). Bar graphs show the means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Transduction, Mutagenesis, Ligand Binding Assay, Expressing, Western Blot, Confocal Microscopy

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Expression levels of EYA2 in human leukemia samples. (A) Expression levels of EYA2 and ACTB in normalized PLZF-RARA APL (n = 5 [GSE8510]) and PML-RARA APL (n = 15 [GSE61804]) samples. (B) Waterfall plot of COPA-transformed expression levels of EYA2 in AML (n = 105 [GSE12662]) samples, including t(15;17) APL (n = 13) samples.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Transformation Assay

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Evaluation of knockdown effects of shRNAs against Eya2. (A) Schematic representation of N-terminally FLAG-tagged Eya2 ligated to a part of the 3′ UTR (FLAG-Eya2/3′UTR) containing target sequences for two shRNAs (shE09 and shE12). The coding sequence of Eya2 except for the first ATG and the partial sequence of the 3′ UTR are shown in thick and thin horizontal lines, respectively. A flag and a black box indicate the FLAG sequence and peptides, respectively. Arrows indicate primers used in RT-qPCR. (B) Experimental strategy for shRNA-mediated Eya2 depletion in PLZF-RARA-immortalized cells forced to express FLAG-Eya2. PLZF-RARA-immortalized cells were retrovirally transduced with FLAG-Eya2/3′ UTR in pMYs-IP. Following puromycin (puro) selection for 3 days, the cells expressing FLAG-Eya2/3′ UTR were next retrovirally transduced with shRNA expressors in pMXsU6-KID. bs, blasticidin; shLuc, shRNA against the luciferase gene. (C and D) Expression levels of Eya2 transcripts by RT-qPCR (C) in comparison with those of FLAG-Eya2 protein by Western blotting analyses (D) in the PLZF-RARA-immortalized cells expressing FLAG-Eya2/3′ UTR after shRNA transduction. Lysates extracted from the shRNA-transduced cells were blotted with anti-FLAG antibody (αFLAG), followed by reprobe with anti-Stat5a as an internal control. Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Sequencing, Quantitative RT-PCR, shRNA, Transduction, Selection, Expressing, Luciferase, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Restoration of clonogenicity of PLZF-RARA-immortalized cells with Eya2 depletion by introduction of shRNA-resistant Eya2. (A) Experimental strategy for analysis of PLZF-RARA-immortalized cells forced to express shRNA-resistant Eya2 with retroviral transduction of shRNA expressors. PLZF-RARA-immortalized cells were retrovirally transduced with the coding sequence alone of Eya2 in pMYs-IP. Following puromycin (puro) selection for 3days, the immortalized cells force to express Eya2 were next retrovirally transduced with shRNA expressors in pMXsU6-KO. (B and C) Expression levels of Eya2 by RT-qPCR (upper panels) and relative CFU (3,000 cells/dish [bottom panels]) of the cells sorted from the shRNA-transduced cells on day 2.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: shRNA, Transduction, Sequencing, Selection, Expressing, Quantitative RT-PCR

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Reduced clonogenicity of Plzf-immortalized and PML-RARA-immortalized cells with Eya2 depletion. (A) Experimental strategy for analysis of Plzf-immortalized cells using pMXsU6-KID. bs, blasticidin. (B and C) Expression levels of Eya2 by RT-qPCR (B) and relative CFU (10,000 cells/dish) (C) of the shRNA-transduced cells. (D) Experimental strategy for analysis of PML-RARA-immortalized cells using pMXsU6-KO. (E and F) Expression levels of Eya2 by RT-qPCR (E) and relative CFU (6,000 cells/dish) (F) of the sorted cells. Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Quantitative RT-PCR, shRNA

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Possible regulation of c-Myc expression by Eya2 was not critically involved in PLZF-RARA-mediated immortalization. (A) Experimental strategy for analysis of PLZF-RARA-immortalized cells using pMXsU6-KID. bs, blasticidin. (B) Expression levels of Eya2 by RT-qPCR in shRNA-transduced cells. (C) Expression levels of total c-Myc and Thr58-phosphorylated c-Myc by Western blotting analyses of PLZF-RARA-immortalized cells with Eya2 depletion. Lysates extracted from the shRNA-transduced cells were blotted with anti-c-Myc (upper), anti-Thr58-phosphorylated c-Myc (p-c-Myc [middle]), and anti-Stat5a antibodies (bottom) as an internal control. Bar graphs show means ± SD from three independent experiments.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing, Quantitative RT-PCR, shRNA, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Characterization of a subtype of AML with high EYA2 expression. (A) COPA revealing EYA2 as a gene with an outlier profile at the 95th percentile in gene expression data for AML samples from TCGA. EYA2 expression is shown in the waterfall plot using the Oncomine web platform. (B) GSEA of AML samples from TCGA showing that gene sets up- and downregulated in leukemic stem cells (LSCs) (GENTLES_LEUKEMIC_STEM_CELL_UP and GENTLES_LEUKEMIC_STEM_CELL_DN) (37) were enriched insignificantly and significantly in clinical samples with high and low levels of EYA2 expression, respectively. (C) GSEA of AML samples (GSE61804) showing that the gene set upregulated in leukemic stem cells (GAL_LEUKEMIC_STEM_CELL_UP) (38) was enriched in samples with high EYA2 expression, compared with those with low expression, using continuous values of EYA2 expression. (D) GSEA of AML samples from TCGA showing that the gene set upregulated in mouse LT-HSCs (IVANOVA_HEMATOPOIESIS_STEM_CELL_LONG_TERM) (39) was enriched in clinical samples expressing high levels of EYA2. NES, normalized enrichment score.

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Expressing

Journal: Molecular and Cellular Biology

Article Title: Eya2, a Target Activated by Plzf, Is Critical for PLZF-RARA -Induced Leukemogenesis

doi: 10.1128/MCB.00585-16

Figure Lengend Snippet: Sequences of primers used in this study

Article Snippet: The prepared samples were boiled, followed by Western blotting analyses using rabbit polyclonal anti-Eya2 (ab116075; Abcam, Cambridge, United Kingdom), anti-RARA, anti-ERα (anti-ER, MC-20), anti-STAT5a (L-20), anti-p-c-Myc (Thr58), goat polyclonal anti-Six1 (A-20), mouse monoclonal anti-c-Myc (C-33), anti-PLZF (D9, Santa Cruz Biotechnology), anti-FLAG, and anti-α-tubulin (Sigma-Aldrich) antibodies.

Techniques: Sequencing