afp goat α mouse notch1 antibody  (Santa Cruz Biotechnology)

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Primers and PCR conditions for respective genes

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Amplification

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Notch1 antisense reduces NOTCH1 expression and NSC self-renewal. NSCs were cultured in the presence of 20 ng/ml EGF in the absence or presence of 20 μmNotch1antisense and harvested after 1 DIV for protein or cultured for a total of 3 DIV (to form P1 neurospheres) and assayed either by single-sphere dissociation (A) or batch culture (B) for the formation of P2 neurospheres.A, Western blot analysis reveals a reduction in NOTCH1-PF1 expression (inset; p < 0.05; t test; n = 3) in antisense-treated P1 neurospheres. A concomitant decrease was observed in the ability of antisense-treated, individual equivalent sized P1 neurospheres to produce P2 neurospheres (*p < 0.05; t test; n = 3) compared with sense treatment. B, Assaying for the ability of P1 neurospheres treated with Notch1 antisense to produce P2 neurospheres by batch culture analysis also reveals a significant decrease in their ability to produce P2 neurospheres (*p < 0.05; t test;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Cell Culture, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Disruption of NOTCH1 signaling by γ-secretase inhibitor II delays P1 neurosphere formation and reduces their ability to produce P2 neurospheres. A, To ensure that the γ-secretase inhibitor that we were using was effectively blocking production of NOTCH1-PF3, NSCs were cultured in 20 ng/ml EGF (20 ng/ml) for 24 hr, at which point DMSO (carrier) or γ-secretase inhibitor II (50 μm) was added, and the cells were harvested 4 hr later for total proteins and Western blot analysis. A, The asterisk indicates an increase in the P2 proteolytic product of NOTCH1, as would be expected if the γ-secretase inhibitor was effectively blocking production of NOTCH1-PF3 (n = 3), and identifies the upper band as furin-processed NOTCH1 or NOTCH1-PF1. B, Three dayin vitro P1 neurospheres that were treated with γ-secretase inhibitor for 4 hr and harvested for nuclear proteins and Western blot analysis demonstrate a decrease in NOTCH1-PF3 compared with DMSO control. C–H, NSCs were cultured in EGF (20 ng/ml) and either DMSO (C, E,G; carrier) or γ-secretase inhibitor II (D, F, H; 30 μm), and digital micrographs were taken after 6 (C, D), 18 (E,F), and 88 hr (G,H). I, Single-sphere dissociation assay reveals a significant reduction in self-renewal capacity of P1 neurospheres generated for 3 DIV in the presence of γ-secretase inhibitor II (30 μm) compared with DMSO controls (*p< 0.05; t test; n = 3).Inset shows a reduction of NOTCH1-PF1 expression in P1 neurospheres treated for 1 DIV, from the time of plating, with 50 μm γ-secretase inhibitor II compared with the DMSO control (p < 0.05; t test;n = 3), indicating that constitutive inhibition of NOTCH1 activation for at least 24 hr leads to an overall decrease in NOTCH1 expression. Scale bar, 100 μm. N.S., Nonspecific; γ-SI, γ-secretase inhibitor.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Blocking Assay, Cell Culture, Western Blot, Generated, Expressing, Inhibition, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: EGF+CNTF treatment of embryonic P1 neurospheres specifically increases Notch1 mRNA and protein expression. A–D, Immunofluorescence micrographs of a coronal section (8 μm) through the forebrain of an E14 mouse embryo.A, Nuclei were labeled with Hoechst 33258 (blue). CNTFRα-immunoreactive cells in the ventricular zone were visualized with Cy3 (B, red), and Notch1-immunoreactive cells were labeled with FITC (C, green). D, A merged image of B and C, where yellow staining indicates colocalization of NOTCH1 and CNTFRα. Box inA indicates area magnified in B–D.E–G, NSCs were cultured in 20 ng/ml EGF, the absence or presence of 20 ng/ml CNTF, and harvested after 24 hr for total RNA and RT-PCR Southern blot analysis (E,F), or after 3 DIV for Western blot analysis (G). Notch1 expression increased significantly (*p < 0.05 vs EGF; ttest; n = 3) after 1 DIV of CNTF treatment (E) compared with no change inNotch3 expression (F). Both the 93-4 and Santa Cruz intracellular NOTCH1 antibodies reveal an increase in NOTCH1-PF1 and NOTCH1-PF2 proteolytic products after 3 DIV of EGF+CNTF treatment compared with EGF alone (G) (p < 0.01; ttest; n = 5). Nuclear expression of NOTCH1-PF3 increases in 3 DIV P1 neurospheres cultured constitutively in EGF+CNTF compared with EGF alone (H) (p < 0.01; t test;n = 4). Scale bars: A, 50 μm;D, 100 μm. LGE, Lateral ganglionic eminence; LV, lateral ventricle; CTX, cortex.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Immunofluorescence, Labeling, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Southern Blot, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: Cell–cell contact is not required for CNTF to increase NOTCH1 expression but is required for CNTF to increase NSC self-renewal. A, Western blot analysis reveals that NOTCH1-PF1 expression increases as early as 2 hr after CNTF treatment of 3 DIV EGF-derived P1 neurospheres (n = 3).B, Totally dissociated primary neurospheres were cultured in EGF+CNTF for 6 hr; Western blot analysis demonstrates a threefold increase in NOTCH1-PF1 expression (p < 0.05; t test;n = 3) compared with EGF controls. No increase in NOTCH1-PF2 could be detected. C, P1 neurospheres were generated in EGF or in EGF+CNTF in the absence (0–24 hr) or presence (72–96 hr) of cell–cell contact. After 7 DIV the three different groups were assayed for the formation of P2 neurospheres by single-sphere dissociation and culture in EGF alone (each group was washed at 24 and 96 hr). Compared with EGF, addition of CNTF for 24 hr at 3 DIV increased the formation of P2 neurospheres by 59% (p < 0.0001; Tukey HSD test;n = 3), whereas there was no difference in P2 neurosphere formation when CNTF was added for the first 24 hr (p > 0.58; Tukey HSD test;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Generated

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF increases the number of intensely NOTCH1-immunoreactive cells. Primary neurospheres were dissociated and then cultured on poly-l-ornithine-coated coverslips for 6 hr in either EGF (A, B) or EGF+CNTF (C, D), and blind counts were made (as described in Materials and Methods) on the number of intensely NOTCH1-immunoreactive cells (C).A, C, Nuclei were labeled using Hoechst 33258 (blue). NOTCH1-immunoreactive cells were labeled with rhodamine (B, D,red). E, Compared with cells cultured in EGF alone, cells cultured in EGF+CNTF demonstrate a 49% increase in the number of intensely NOTCH1-immunoreactive cells (*p < 0.003; t test;n = 3). Arrowheads indicate examples of cells that stain intensely for NOTCH1, and small arrows indicate examples of cells that stain weakly for NOTCH1. Scale bar, 20 μm.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Cell Culture, Labeling, Staining

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: IL6+sIL6R increases NOTCH1 expression and P2 neurosphere production in P1 neurospheres generated fromLIFRβ−/−mice. P1 neurospheres were generated from wild-type (+/+) or null mutant (−/−) LIFRβ littermates, in the various conditions indicated, and were then assayed after 3 DIV for NOTCH1 protein with Western blot and after 7 DIV for P2 neurosphere production by single-sphere dissociation in EGF alone. Increase in P2 neurosphere production in wild-type (+/+) EGF+CNTF generated P1 neurospheres and inLIFRβ−/−P1 neurospheres generated in EGF+IL6+sIl6R correlated with concomitant increases in NOTCH1-PF1 expression (inset). CNTF had no effect on P2 neurosphere production or NOTCH1-PF1 expression inLIFRβ−/−P1 neurospheres. **p < 0.01 versus +/+ control culture or −/− control culture; Tukey HSD test; n= 5. N-PF1, NOTCH1-PF1.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Generated, Mutagenesis, Western Blot

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: The CNTF-induced increase in NOTCH1 expression in dissociated primary neurospheres is dependent on either EGF or FGF2 signaling. A, B, Single-cell suspensions derived from primary neurospheres and cultured for 6 hr in the indicated conditions reveal that CNTF had no effect on NOTCH1-PF1 expression in the absence of EGF (B) (n = 3) and that CNTF can increase NOTCH1-PF1 expression in either EGF- or FGF2-containing media (B) (p< 0.05; t test; n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Derivative Assay, Cell Culture

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF enhances the expression of NOTCH1 in vivo. Adult CD1 mice were infused with either EGF (A, C, D) or EGF+CNTF (B, E, F) for 6 d, after which brains were processed for NOTCH1 immunohistochemistry. Infusion of EGF+CNTF resulted in an overall increase in NOTCH1 staining intensity as well as a markedly thickened layer of NOTCH1 expression on the lateral aspect (A, B, arrows) of the ventricle. Furthermore, more cells in the ventricular zone labeled for NOTCH1 in EGF+CNTF (C, NOTCH1, 76 ± 12%;D, Hoechst) compared with EGF (E, NOTCH1, 39 ± 7%; F, Hoechst) infused mice (p < 0.026; t test;n = 3 each group). C–F,Arrows indicate NOTCH1 unlabeled cells. Scale bars:B, 100 μm; F, 25 μm.

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, In Vivo, Immunohistochemistry, Staining, Labeling

Journal: The Journal of Neuroscience

Article Title: Glycoprotein 130 Signaling Regulates Notch1 Expression and Activation in the Self-Renewal of Mammalian Forebrain Neural Stem Cells

doi: 10.1523/JNEUROSCI.23-05-01730.2003

Figure Lengend Snippet: CNTF treatment changes the expression of genes regulated by NOTCH1 signaling. A–C, Primary neurospheres derived from the E14 striatum were grown in the presence of EGF for 7 DIV, dissociated, and then cultured (5 × 104 cells/ml) in either EGF or EGF+CNTF. The cells were then harvested for total RNA or protein at 3 DIV and processed for RT-PCR Southern or Western blot analyses as described in Materials and Methods. A, Constitutive CNTF treatment significantly decreases Hes5 expression, whereas Hes1expression in CNTF-treated P1 neurospheres does not differ significantly from 3 DIV EGF-derived P1 neurospheres. B,Mash1 expression, mRNA, and protein are significantly reduced in P1 neurospheres cultured for 3 DIV in the presence of EGF+CNTF compared with EGF alone. C,Delta3 expression is reduced in 3 DIV EGF+CNTF P1 neurosphere cultures compared with EGF cultures. *p< 0.05 versus EGF; t test (A–C;n = 3).

Article Snippet: Nitrocellulose membranes were incubated with the 93-4 rabbit α mouse NOTCH1 primary antibody (1:10,000), or affinity-purified (AFP) goat α mouse NOTCH1 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), or mouse α mouse MASH1 (1:25; gift from Dr. David Anderson, California Institute of Technology), and/or AFP goat α ACTIN (1:100; Santa Cruz Biotechnology) mouse overnight in the blocking buffer at 4°C, washed with Tris-buffered saline (0.1% Tween 20), and then incubated with blocking buffer plus the appropriate secondary antibody conjugated to horseradish peroxidase (Chemicon, Temecula, CA).

Techniques: Expressing, Derivative Assay, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Western Blot