mouse anti klf4 mab  (Santa Cruz Biotechnology)

Journal: Blood

Article Title: NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis

doi: 10.1182/blood-2010-07-295261

Figure Lengend Snippet: NANOG and KLF4 both bind to the NANOG promoter/enhancer and NANOG binds to the FLK1 promoter/enhancer in response to WNT3A. (A) HUVECs were starved of growth factors and serum, then left untreated or treated for 120 minutes with WNT3A (50 ng/mL). ChIP was performed with antibodies (Abs) specific for NANOG and KLF4, in addition to a nonspecific anti-rabbit IgG, as indicated (left). The left panel shows the PCR products (2.0 kb) from the human NANOG promoter/enhancer with the use of input chromatin prepared from HUVECs treated with or without WNT3A. (B) The primers used for amplification of the human NANOG promoter/enhancer region. (C) The relative positions of the primers used for amplification of the human FLK1 promoter region flanking the 8 putative NANOG binding sites. (D) Primers used for amplification of the human FLK1 promoter. (E) Interaction of NANOG with the FLK1 promoter. PCR product of the FLK1 promoter/enhancer (1.0 kb). (F) Biotinylated probes used for the EMSA. (G) Representative images of the EMSA experiment. Biotin-labeled oligonucleotides (probe 1 and probe 2) containing the sequences of the putative NANOG binding sites from the FLK1 promoter were incubated with nuclear extracts. The supershift was carried out by preincubation of the nuclear extracts with an anti-NANOG antibody. Note: Both monomeric and dimeric forms of NANOG interacted with the biotinylated probes. Arrowheads indicate the dimeric forms of NANOG in a native gel. Experiments were carried out ≥ 5 times.

Article Snippet: Antibodies and reagents The mouse anti–human NANOG monoclonal antibody (mAb) and mouse anti–human KLF4 mAb were purchased from Abnova.

Techniques: Amplification, Binding Assay, Labeling, Incubation

Journal: Blood

Article Title: NANOG induction of fetal liver kinase-1 (FLK1) transcription regulates endothelial cell proliferation and angiogenesis

doi: 10.1182/blood-2010-07-295261

Figure Lengend Snippet: NANOG knockdown decreases angiogenesis in Matrigel plug assays. (A) Timeline of the Matrigel plug assay. (B-D) HUVECs were incubated with the indicated shRNAs (retrovirus) for 10 hours (in presence of serum and growth factors), after which time the cells (105) were mixed with growth facto–reduced Matrigel (250 μL) plus WNT3A (50 ng/mL) and injected subcutaneously into athymic nude mice (n = 5). After 7 days, the Matrigel plugs were collected, embedded in paraffin, sectioned (4-5 μm), and stained with hematoxylin and eosin. Pictures of Matrigel plugs were taken using a Canon Powershot A640 digital camera controlled by Zoom Browser Ex software 5.7. Digital images were then saved as EPS documents using Adobe Photoshop CS. Multiple images were assembled using QuarkXpress 8.0 software and labeled, and final images were saved as EPS documents. (E-G) Note the prominent microvessels in the control shRNA (noninterfering shRNA retrovirus) plugs and the reduced number of capillaries in the NANOG knockdown plugs. FLK1 cDNA expression partially compensated for the loss of NANOG. Scale bar, 40 μm. (H) Quantification of microvessel capillaries in the Matrigel plugs. The data represent the mean ± SEM; n = 5-10 from 3-5 independent experiments; *P < .05 and **P < .01 compared with control or as indicated. (I-K) Thin cryosections (5 μm) prepared from Matrigel plugs were stained with anti–human von Willebrand Factor (red) or anti–mouse CD31(green) to distinguish human and mouse ECs. Highly autofluorescent red blood cells inside neovessels are yellow in color. Note that close association between human and murine ECs are indicated by white arrows. Green arrows indicate HUVECs that failed to form blood vessels; yellow arrow indicates neovessel made of HUVECs. Original magnification, ×200. Epifluorescence images were captured using a Zeiss Axioplan 2 inverted microscope under 20× objectives, using an AxioCam H digital camera. Digital images were saved as TIFF documents using Adobe Photoshp CS. Multiple images were assembled using QuarkXpress 8.0 software and labeled, and final images were saved as EPS documents.

Article Snippet: Antibodies and reagents The mouse anti–human NANOG monoclonal antibody (mAb) and mouse anti–human KLF4 mAb were purchased from Abnova.

Techniques: Knockdown, Matrigel Assay, Incubation, Injection, Staining, Software, Labeling, Control, shRNA, Expressing, Inverted Microscopy

Journal:

Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

doi: 10.1161/CIRCRESAHA.110.219592

Figure Lengend Snippet: A-F, Untreated or ECs treated with LiCl (20 ng/mL) or with recombinant WNT3A (50 ng/mL) for 3 d and stained for VE-cadherin. Representative images of control and treated ECs at 200× (A, C, E) and 400× (B, D, F) magnification. For additional images, see Figure S2. G-J) Cell extracts were analyzed by Western blot (WB) for VE-cadherin, β-catenin, KLF4, and GAPDH. Results are representative of at least three separate experiments.

Article Snippet: Mouse anti-human KLF4 mAb (H00009314-M01) was purchased from AbNOVA (Walnut, CA).

Techniques: Recombinant, Staining, Control, Western Blot

Journal:

Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

doi: 10.1161/CIRCRESAHA.110.219592

Figure Lengend Snippet: A, Timeline of TER assay. B, HLMVECs plated on gold microelectrodes were left untreated (control) or transfected with a non-silencing siRNA or KLF4-silencing siRNA, followed by TER assay. Note that KLF4 silencing inhibited the thrombin response relative to untreated group (no transfection) or negative control group (non-silencing siRNA; n = 5 per group). Mean value (± s.e.m.) of maximal TER responses to thrombin (50 nM) stimulation (n = 7). Thrombin-induced decrease in TER was significantly attenuated in HLMVECs transfected by KLF4-depletion compared with untreated control or negative control group transfected with a non-silencing siRNA. C-E, KLF4 knockdown increases transendothelial permeability of fluorescein isothiocyanate (FITC)-conjugated albumin by decreasing VE-cadherin expression and AJ integrity. C, Timeline of experiments. D, Confluent HLMVEC monolayers were grown on microporous filters for 36 h, either left alone (control) or treated with control siRNA or with KLF4-siRNA for 12 hours. At 18 hr post transfection, transendothelial FITC-albumin permeability was measured. Control HLMVECs showed basal transendothelial FITC-albumin permeability values, while KLF4 knockdown increased transendothelial FITC-albumin permeability. Re-expression of VE-cadherin into KLF4-depleted ECs partially restored the effect of loss of KLF4. Values are mean ± s.e.m. (n= 10). *p < 0.05 vs other control (untreated) group. **p <0.01 KLF4 siRNA vs control siRNA. E, The efficiency of KLF4-knockdown and VE-cadherin re-expression in HLMVECs was determined by Western blotting.

Article Snippet: Mouse anti-human KLF4 mAb (H00009314-M01) was purchased from AbNOVA (Walnut, CA).

Techniques: Control, Transfection, Negative Control, Knockdown, Permeability, Expressing, Western Blot

Journal:

Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

doi: 10.1161/CIRCRESAHA.110.219592

Figure Lengend Snippet: A, Schematic of human VE-cadherin promoter −1.3 kb upstream of transcription start site (TSS). Potential KLF4 binding (CACCC) sites are indicated. B, Sequence of human VE-cadherin primer pair used for ChIP experiments. C, HLMVECs and HUVECs were grown in complete media, and left untreated (-) or treated for 3 days with WNT3A. ChIP assay was performed with indicated antibodies. PCR product of VE-cadherin promoter using input chromatin. D, Biotin-labeled oligonucleotide probes (P1-P4) containing CACCC sites used for EMSA. E, Representative image of EMSA blot. Probe-1 (P1) of KLF4 site from VE-cadherin-promoter was incubated with nuclear extracts prepared from ECs treated with WNT3A or by pre-incubation of nuclear extract with anti-KLF4 antibody in the presence or absence of cold-unlabeled oligonucleotide. Results are representative of at least three separate experiments.

Article Snippet: Mouse anti-human KLF4 mAb (H00009314-M01) was purchased from AbNOVA (Walnut, CA).

Techniques: Binding Assay, Sequencing, Labeling, Incubation

Journal:

Article Title: Kr?ppel-Like Factor-4 Transcriptionally Regulates VE-cadherin Expression and Endothelial Barrier Function

doi: 10.1161/CIRCRESAHA.110.219592

Figure Lengend Snippet: A, Time-line of siRNA administration, LPS challenge, myeloperoxidase (MPO) as a measure of lung neutrophil sequestration, and lung extravascular water content assays. B, Lung MPO activities were assayed in mice receiving either control siRNA or Klf4-specific siRNA with or without LPS challenge at 20 min, 1 h, 3 h, and 6 h. C, Lung tissue extracts were prepared 18 h after siRNA administration (but prior to receiving LPS) and efficacy of Klf4-knockdown in lung tissues was evaluated by immunoblotting with the indicated antibodies. Values are mean ± s.e.m. (n=12 per group). *p < 0.01 vs control (untreated) group. **p <0.05 Klf4 siRNA vs control siRNA.

Article Snippet: Mouse anti-human KLF4 mAb (H00009314-M01) was purchased from AbNOVA (Walnut, CA).

Techniques: Control, Knockdown, Western Blot