Anti-Nucleophosmin (phospho T199) antibody [EP1857Y]

• ICC/IF

AbReview15450****

Immunocytochemistry/ Immunofluorescence - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

ab81551 staining Nucleophosmin (phospho T199) in human HeLa cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed with formaldehyde, permeabilized with 0.1% Triton x100 in PBS and blocking with 5% serum was performed for 30 minutes at 23⁰C. Samples were incubated with primary antibody (1/150 in PBS) for 1 hour at 23°C. An Alexa Fluor^®488-conjugated goat polyclonal to rabbit IgG was used at dilution at 1/100 as secondary antibody.

This image is courtesy of an anonymous Abreview.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

Immunohistochemistry analysis of paraffin-embedded Human lymphoma tissue, using 1/100 ab81551.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

Immunofluorescent staing of HeLa cells using 1/100 ab81551.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

Overlay histogram showing HeLa cells stained with ab76539 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab76539, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• WB

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Western blot - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

All lanes:

Western blot - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (ab81551) at 1/2500 dilution

Lane 1:

lysate from untreated HeLa cells at 10 µg

Lane 2:

Lysate from HeLa cells serum-starved overnight, and then treated with 100ng/ml Calyculin A for 30min at 37℃ at 10 µg

Secondary

All lanes:

HRP labelled Goat anti-Rabbit at 1/2000 dilution

Predicted band size: 32 kDa

Observed band size: 33 kDa

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• WB

CiteAb

Western blot - Anti-Nucleophosmin (phospho T199) antibody [EP1857Y] (AB81551)

Nucleophosmin (phospho T199) western blot using anti-Nucleophosmin (phospho T199) antibody [EP1857Y] ab81551. Publication image and figure legend from Mukhopadhyay, A., Sehgal, L., et al., 2016, Sci Rep, PubMed 27253419.

ab81551 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 ab81551 please see the product overview.

Increased NPM1 phosphorylation at T199 leads to centrosome duplication in 14-3-3γknockdown cells.(a) Protein extracts from vector control or 14-3-3γ knockdown cells were resolved on SDS-PAGE followed by immuno-blot with the indicated antibodies. Actin served as loading control. (b,c) HCT116 cells were transfected with mCherry-α-tubulin and co-transfected with either the control plasmid (pCDNA3) or plasmids encoding wild type cdk1 (cdk1) or constitutively active cdk1 (cdk1AF). Post-transfection the cells were stained with antibodies to Cep170 (green) and DAPI (blue) (b). The percentage of mitotic cells with >2 centrosomes was determined in three independent experiments (c). (d) Protein extracts from the vector control and 14-3-3γ knockdown cells were resolved by SDS-PAGE, followed by Western blot with the indicated antibodies. Note that NPM1 is phosphorylated on T199 to a greater degree in the 14-3-3γ knockdown cells. Actin served as loading control. (e) The 14-3-3γ -knockdown and vector control cells were transfected with either the vector control or FLAG-epitope tagged versions of WT NPM1 or the NPM1 mutants (T199A and T199D). Post transfections, protein extracts prepared from these cells were resolved by SDS-PAGE followed by Western blot with the indicated antibodies. Western blots for actin serves as loading controls. (f,g) 14-3-3γ-knockdown and vector-control cells, transfected with mCherry-α-tubulin and FLAG-NPM1 constructs, were stained with antibodies to Cep-170, co-stained with DAPI and followed by confocal microscopy (f) to determine the percentage of cells containing >2 centrosomes. The mean and standard deviation of three independent cells is plotted (g) p values were determined using a Student's t-test (2 sample unequal variance) with p < 0.05. Scale bars indicate 5 μm. All the Western blots were run under the same experimental conditions and the full length blots are in Supplementary Fig. 6.

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