Anti-Neurofibromin antibody

• IP

Supplier Data

Immunoprecipitation - Anti-Neurofibromin antibody (AB17963)

Whole cell lysate (0.5-1.0mg per IP reaction; 20% of IP loaded) from HeLa cells prepared using NETN lysis buffer.

Lane 1 : IP using rabbit anti-Neurofibromin antibody.
Lane 2 : IP using ab17963 at 6 μg per reaction.
Lane 3 : Control IgG.

For western blotting, ab17963 was used at 1 μg/ml.

All lanes:

Immunoprecipitation - Anti-Neurofibromin antibody (ab17963)

Predicted band size: 319 kDa

true

Exposure time: 30s

• WB

Supplier Data

Western blot - Anti-Neurofibromin antibody (AB17963)

All lanes:

Western blot - Anti-Neurofibromin antibody (ab17963) at 0.1 µg/mL

Lane 1:

HeLa whole cell lysate in NETN lysis buffer at 50 µg

Lane 2:

293T whole cell lysate in NETN lysis buffer at 50 µg

Lane 3:

Jurkat whole cell lysate in NETN lysis buffer at 50 µg

Lane 4:

TCMK-1 whole cell lysate in NETN lysis buffer at 50 µg

Lane 5:

NIH3T3 whole cell lysate in NETN lysis buffer at 50 µg

Predicted band size: 319 kDa

true

Exposure time: 75s

• WB

CiteAb

Western blot - Anti-Neurofibromin antibody (AB17963)

Neurofibromin western blot using anti-Neurofibromin antibody ab17963. Publication image and figure legend from Yzaguirre, A. D., Padmanabhan, A., et al., 2015, Elife, PubMed 26460546.

ab17963 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab17963 please see the product overview.

Neurofibromin protein expression and activity from the Nf1 alleles.(A) Total cell lysates from E10.5 Nf1+/+, Nf1+/-, Nf1-/-, Nf1GRD/+, and Nf1GRD/GRD embryos were analyzed by SDS-PAGE followed by immunoblot with either anti-neurofibromin (top panel) or anti-beta tubulin (bottom panel) antibodies as indicated. (B) Band intensities from 5 immunoblots as in (A) were quantified by ImageJ. The relative neurofibromin expression for each genotype compared to wild-type is indicated. All data are represented as the mean ± S.E. **, p<0.05; ***, p<0.001; NS = not significant (p<0.001, one-way ANOVA between groups, post hoc multiple comparisons, Tukey's test). (C) A cross-section of a peripheral nerve (demarcated in white and indicated by an arrow) from each of Nf1GRD/flox and Wnt1-Cre; Nf1GRD/flox P0 animals shows elevated expression of pERK, a downstream indicator of Ras activity, in Wnt1-Cre; Nf1GRD/flox animals (right panel). An adjacent blood vessel (BV) is indicated. (D) Adrenal medullary tissue within an adrenal gland from either a Nf1GRD/flox or Wnt1-Cre; Nf1GRD/flox animal shows increased pERK expression in a hyperplastic area from the Wnt1-Cre; Nf1GRD/flox animal (right panel). pERK-positive cells are marked by arrowheads. Background fluorescence from non-neural-crest-derived adrenal cortical and blood cells is evident in the Nf1GRD/flox sample. (E) Cardiac cushions from E11.5 embryos show elevated pERK staining in Nf1GRD/GRD embryos compared to Nf1+/+ animals as indicated within the dashed oval. Scale bars = 50 μm.DOI : http : //dx.doi.org/10.7554/eLife.07780.007Generation of Nf1GRDand Nf1GRDCTL mouse lines.(A) Schematic diagram outlining the targeting strategy to develop the Nf1GRD mouse line by modifying the endogenous mouse Nf1 locus with a mutation corresponding to the human NF1 R1276P missense allele. This mutation abrogates neurofibromin GAP activity without impairing secondary or tertiary protein structure or reducing cellular levels of neurofibromin (Klose, et al., 1998). (B) Strategy to develop the Nf1GRDCTLknock-in 'control' mouse by targeting the endogenous mouse Nf1 locus with a construct identical to that used to target the NF1 R1276P mutation in (A) with the exception that no mutation is introduced. Knock-in Nf1GRDCTLmice generated from this construct are a stringent control for Nf1GRDanimals. For both (A) and (B) asterisks denote regions where additional DNA sequences are identically introduced into introns as part of the targeting process. The 'an' cassette imparts G418 resistance and is self-excised in the male germ line. N = NcoI restriction endonuclease site. (C) Southern blots of genomic DNA from embryonic stem (ES) cell clones targeted with either the Nf1GRDor Nf1GRDCTLallele display a 13 kb wild type (WT) band as well as a 5.5 kb mutant (MT) band. Five and three positive clones were isolated with genotype Nf1GRDor Nf1GRDCTL, respectively, as shown. (D) DNA products from PCR reactions performed with primers specific for the Nf1 wildtype, Nf1 knockout (KO), or Nf1 GRD alleles using template DNA isolated from amniotic sacs of E10.5 embryos.DOI : http : //dx.doi.org/10.7554/eLife.07780.008Increased pERK staining in neural crest derivatives of Nf1GRD/flox newborn animals following deletion by Wnt1-Cre.(A) pERK staining was observed in neural crest-derived enteric ganglia within the intestinal wall that was more evident in Wnt1-Cre; Nf1GRD/flox animals (right panel). Arrowheads indicate cells exhibiting positive staining. (B) Both axons (arrows) and nerve cell bodies (arrowheads) were readily visualized in Wnt1-Cre; Nf1GRD/flox newborns but not in control animals (data not shown).DOI : http : //dx.doi.org/10.7554/eLife.07780.009

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