goat polyclonal anti scf  (R&D Systems)

Journal: Blood

Article Title: KIT signaling is dispensable for human mast cell progenitor development

doi: 10.1182/blood-2017-03-773374

Figure Lengend Snippet: SCF is dispensable for mast cell progenitor survival in vitro. CD34+ progenitors were purified from buffy coats from healthy donors and analyzed with flow cytometry. (A-B) The representative gating strategy and the quantification of CD34+CD117int/hiFcεRI+ mast cell progenitors are shown (n = 9). The representative example in panel A is indicated with an open circle. The line in panel B represents the geometric mean. (C-G) CD34-enriched cells were cultured and analyzed by flow cytometry. (D) The fraction of CD117hiFcεRI+ pre–mast cells from panel C was normalized to the combined IL-3 and IL-6 condition for each buffy coat, and the results from 3 buffy coats were pooled. The bars represent the means ± standard error of the mean (SEM). (E) CD34+ progenitors were cultured with IL-3 and IL-6, and the fractions of CD34-expressing cells out of the CD117hiFcεRI+ mast cell population (days 3-7) are shown. The CD34 expression of CD117int/hiFcεRI+ cells is shown as the day 0 control. The bars represent the means ± SEM of 3 buffy coats. (F) The CD117 expression of CD34+ cells was analyzed on day 3. The red histogram indicates the cells cultured without cytokines. The blue line indicates the cells cultured with IL-3 and IL-6. The dashed line indicates the cells cultured with IL-3, IL-6, and SCF. One representative experiment out of 3 is shown. (G) The CD117 expression on CD34+ cells at day 3, shown in panel F, was quantified by calculation of the median fluorescence intensity. The expression level of CD117 is shown as a percentage of the IL-3 and IL-6 condition. The bars represent the means ± SEM of 3 buffy coats. (H-J) CD34+ cells were enriched from buffy coats, cultured, and analyzed by flow cytometry. The medium was supplemented with polyclonal goat IgG or anti-SCF neutralizing antibodies as indicated. (J) The integrin β7 expression was analyzed by flow cytometry before and after culture with IL-3, IL-6, and anti-SCF. The bars in panels I and J represent the means ± SEM of 3 buffy coats. Live singlet cells are shown in the flow cytometric graphs. The statistical analyses in panels D, E, G, and I were performed using 1-way ANOVA with Tukey’s multiple comparisons test. **Adjusted P < .01; ****adjusted P < .0001; ns = not significant. The unpaired 2-tailed Student t test was used for the statistical analysis in panel J. ****P < .0001.

Article Snippet: Polyclonal goat anti-human SCF or polyclonal goat IgG (5 μg/mL; both from PeproTech) was added to the culture medium in some experiments.

Techniques: In Vitro, Purification, Flow Cytometry, Cell Culture, Expressing, Control, Fluorescence

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , KIT Y721 phosphorylation was activated by exogenous rhKITLG (100 ng/mL for 10 min following 2 h serum deprivation [ , ]) in GIST-T1 cells containing a heterozygous activating KIT mutation (n=4/group) and in GIST-T1-5R cells containing an additional, imatinib-resistant KIT mutation (n=6/group), but not in GIST882 cells lacking a WT KIT allele (n=5/group). B , Culturing with anti-human KITLG neutralizing antibody for 4 days inhibited baseline proliferation of GIST-T1 cells ( P <0.001; n=3; regression and 95% confidence band are shown as solid and dashed lines, respectively) and GIST-T1-5R cells ( P <0.001; n=6). The effect of KITLG immunoneutralization on GIST-T1 cells was blocked by preabsorbing the antibody with 10-fold molar excess of rhKITLG (see open symbols in the left panel, second row; n=3/group). KITLG immunoneutralization did not inhibit the proliferation of GIST882 or GIST48B cells lacking a WT KIT allele (note that GIST48B cells also express very low to undetectable levels of KIT protein, see ) and of LX-2 cells lacking KIT protein (n=3/cell line). C , Inhibition of the proliferation of GIST-T1 and GIST-T1-5R cells by siRNA-mediated knock-down of KITLG (n=4/cell line/group; P GIST-T1 : day 2: 0.008, days 4, 6 and 8: <0.001; P GIST-T1-5R : day 2: <0.02, day 4: 0.004, days 6 and 8: <0.001).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Phospho-proteomics, Mutagenesis, Inhibition, Knockdown

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: Results obtained in murine and human smooth muscle cells ( A - E ), human LX-2 stellate cells ( F - G ) and human GIST-T1 cells ( H - I ) are shown. A , Hoffman modulation contrast image of primary human gastric smooth muscle cells. B , KITLG mRNA (total: soluble+membrane-bound) was readily detectable in primary human smooth muscle cells (passage 3) maintained with Smooth Muscle Growth Medium-2 containing insulin, hFGF-B, hEGF and 5% FBS (Lonza) but not in 24-h growth factor- and serum-deficient basal medium. C , Both IGF1 (100 ng/mL) and the GSK3α/β inhibitor SB415286 (30 µM) stimulated Kitl expression in murine gastric tunica muscularis organotypic cultures (n=3/group). D - E , SB415286 stimulated endogenous Kitl expression in murine primary gastric smooth muscle cells ( D ; n=3/group) and KITLG transcriptional activity in the same cell type transfected with a KITLG promoter (-2120 bp to +407 bp)-pGL3 luciferase construct ( E ; n=3/group). IGF1 (100 ng/mL; n=3/group) and SB415286 (30 µM; n=6/group) also increased endogenous KITLG mRNA expression in LX-2 ( F ) and GIST-T1 cells ( H ) and stimulated KITLG promoter activity in a time-dependent fashion (LX-2: n=6-9/group; G ; GIST-T1: n=3-11/group; I ). Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other ( P <0.05 by post-hoc multiple comparisons).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Membrane, Expressing, Activity Assay, Transfection, Luciferase, Construct, Control

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , Increased occupancy of the Kitl core promoter region by H4ac in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. Only SB415286 increased occupancy by H3K9ac ( B ). Representatives of two independent experiments, each performed in triplicates, are shown. C , Dose-dependent stimulation of KITLG expression in LX-2 cells by 24-h treatment with the class I-II HDAC inhibitor SAHA (n=3/group). D , Dose-dependent inhibition of the SB415286-induced stimulation of KITLG expression by the p300/PCAF HAT inhibitor garcinol (24 h) in LX-2 cells (n=3/group). Drugs were applied following 24-h serum deprivation. Groups marked by asterisk are different from the control group, and groups not sharing the same superscript letter are different from each other ( P <0.05 by post-hoc multiple comparisons).

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Muscles, Expressing, Inhibition, Control

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A , Increased occupancy of the Kitl core promoter by the trithorax group-mediated, activating H3K4me2 histone modification in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. B - C , Reduced occupancy of the Kitl core promoter by the PRC2-mediated, repressive H3K27me3 histone modification ( B ) and by the PRC2 histone methyltransferase EZH2 ( C ) in response to rhIGF1 and SB415286 in the same tissues. D - F , Stimulation of KITLG expression by the indirect histone methyltransferase inhibitor Adox in murine gastric smooth muscles ( D ), LX-2 cells ( E ) and GIST-T1 cells ( F ) (n=3/group). Adox was applied for 72 h following 24-h serum deprivation. See further details in the legend to .

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Modification, Muscles, Expressing

Journal: PLoS ONE

Article Title: Membrane-To-Nucleus Signaling Links Insulin-Like Growth Factor-1- and Stem Cell Factor-Activated Pathways

doi: 10.1371/journal.pone.0076822

Figure Lengend Snippet: A - B , Reduced occupancy of the Kitl core promoter by the repressive H3K9me2 ( A ) and H3K9me3 ( B ) histone modifications in response to 6-h rhIGF1 (100 ng/mL) and SB415286 (30 µM) treatment in murine gastric smooth muscles. C - D , Probing the role of HP1 isoforms in transcriptional repression of KITLG in LX-2 cells by siRNA- (CBX5: HP1α; 25 nM, 72 h; C ) or shRNA-mediated knock-down (CBX1: HP1β; CBX3: HP1γ; 30 µg plasmid, 72 h; D ). Note activation of KITLG expression by shRNA-mediated knock-down of HP1γ (n=3/group). E - F , Stimulation of KITLG expression by the G9A/GLP H3K9me1/2 HKMT inhibitor BIX-01294 in LX-2 cells ( E ) and GIST-T1 cells ( F ) (n=3/group). G , Stimulation of KITLG expression by the H3K9 HKMT inhibitor chaetocin in LX-2 cells (n=3/group). Drugs were applied for 24 h following 24-h serum deprivation. See further details in the legend to .

Article Snippet: The role of endogenous KITLG in the proliferation of GIST-T1, GIST-T1-5R, GIST882, GIST48B and LX-2 cells was tested by culturing in the above media in the presence of purified, azide-free goat polyclonal anti-human KITLG antibody (AB-255-NA, R&D Systems, Minneapolis, MN; applied for 4 days at concentrations indicated in the Results), which has been shown to neutralize KITLG-induced proliferation in the TF1 human erythroleukemic cell line [ ].

Techniques: Muscles, shRNA, Knockdown, Plasmid Preparation, Activation Assay, Expressing