Anti-Nucleolin antibody [364-5]

• ICC/IF

AbReview41541****

Immunocytochemistry/ Immunofluorescence - Anti-Nucleolin antibody [364-5] (AB136649)

ab136649 staining Nucleolin in human breast cell line by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 5% Triton X-100 in PBS and blocked with 5% BSA for 1 hour at 25°C. Samples were incubated with primary antibody (1/1000) for 1 hour. An undiluted Alexa Fluor® 546-conjugated anti-mouse IgG polyclonal was used as the secondary antibody.

This image is courtesy of an anonymous Abreview

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nucleolin antibody [364-5] (AB136649)

Immunocytochemical analysis of HDFn cells (paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min)) labelling Nucleolin with ab136649 at 1µg/ml (red) and anti-GAPDH (ab110305) in green, as a counter stain. Secondary : Alexa 594 goat anti-mouse IgG1 at a 1/1000 dilution.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Nucleolin antibody [364-5] (AB136649)

Immunocytochemical analysis of HeLa cells (paraformaldehyde fixed (4%, 20 min) and Triton X-100 permeabilized (0.1%, 15 min)) labelling Nucleolin with ab136649 at 1μg/ml (red) and anti-GAPDH (ab110305) in green, as a counter stain.
Secondary : Alexa 594 goat anti-mouse IgG1 at a 1/1000 dilution.

• Flow Cyt

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Flow Cytometry - Anti-Nucleolin antibody [364-5] (AB136649)

Flow Cytometric analysis of HeLa cells (fixed and permeabilized with methanol) labelling Nucleolin with ab136649 (blue) at 1µg/ml or an equal amount of an isotype control antibody (red).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nucleolin antibody [364-5] (AB136649)

IHC image of nucleolin staining in human liver HCC formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab136649, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IP

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Immunoprecipitation - Anti-Nucleolin antibody [364-5] (AB136649)

Detection of Nucleolin by Immunoprecipitation.
Nucleolin in HepG2 cell lysate (1 mg) was immunoprecipitated using 1 µg ab136649 antibody. The gel was stained with Coomassie brilliant blue G.

All lanes:

Immunoprecipitation - Anti-Nucleolin antibody [364-5] (ab136649)

Predicted band size: 76 kDa

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• WB

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Western blot - Anti-Nucleolin antibody [364-5] (AB136649)

All lanes:

Western blot - Anti-Nucleolin antibody [364-5] (ab136649) at 1 µg/mL

Lane 1:

HepG2 whole cell lysate at 20 µg

Lane 2:

HDFn whole cell lysate at 25 µg

Lane 3:

SH-SY5Y whole cell lysate at 20 µg

Secondary

All lanes:

Goat polyclonal to Mouse IgG - AP at 1/3000 dilution

Predicted band size: 76 kDa

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• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Nucleolin antibody [364-5] (AB136649)

Immunocytochemistry-immunofluorescence using Anti-Nucleolin antibody [364-5], ab136649. Publication image from Dubois, M. L. et al., 2020, Nat Commun, 32161257. Legend direct from paper.

UbKEKS is important for cell growth and regulates lamin A localization.a–b Cells were transfected with either GFP-tagged lamin A (LMNA) or lamin B2 (LMNB2), and with HA-tagged Ub or UbKEKS. Total cell lysates (Extract) or IPs with GFP-Trap were loaded on SDS-PAGE and revealed with GFP or HA antibodies (n = 3 independent experiments). c Knockout of UBBP4 was performed using CRISPR/Cas9 and two different combinations of guide RNAs in HeLa cells. d Cells are sorted from the debris according to FSC-A and SSC-A, and then single cells are selected using FSC-H and FSC-W for measuring using the FITC channel. Wild type and KO HeLa cells (clones 2.7 and 4.3) were assessed for growth using a CFSE assay at each time points for 96 h. Data are presented as mean values with ±SD. Statistical analysis was performed using two-way ANOVA (F(2,84) = 45.70) and Dunnett’s multiple comparison post hoc tests (n = 8 independent experiments). e Wild type and KO HeLa cells (clones 2.7 and 4.3) were labeled for immunofluorescence microscopy with a lamin A antibody. The nuclei were stained with DAPI (n = 3 independent experiments). f Control and KO HeLa cells (clones 2.7 and 4.3) were labeled for immunofluorescence microscopy with a Nucleolin antibody. The nuclei were stained with DAPI (n = 3 independent experiments). g Quantification of nucleoli was achieved using CellProfiler, through a primary object identification using DAPI and Nucleolin staining, respectively. Each nucleolus was reported to the parental nucleus and the area was determined by measuring the object size shape. Data are presented as box plots where the center lines show the medians; box limits indicate the 25th and 75th percentiles as determined by Prism software; whiskers extend to minimum and maximum values; individual data points are superimposed on the box plot. Statistical analysis was performed using two-way ANOVA (F(2,540) = 37.08) and Dunnett’s multiple comparison post hoc tests (n = 90 cells examined over three independent experiments). Source data are provided as a Source Data file.

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-Nucleolin antibody [364-5] (AB136649)

Immunocytochemistry-immunofluorescence using Anti-Nucleolin antibody [364-5], ab136649. Publication image from Nihei, Y. et al., 2020, Acta Neuropathol, 31642962. Legend direct from paper.

Reduction of hnRNPA3 increases RNA foci number. Knockdown of hnRNPA3 increases antisense RNA foci in fibroblasts derived from C9orf72 mutation carriers. a Western blotting with anti-hnRNPA3, hnRNPA1, and hnRNPA2B1 antibodies confirms selective and efficient siRNA-mediated knockdown of hnRNPA3. β-actin serves as a loading control. b Fluorescent in situ hybridization (FISH) of antisense repeat RNA foci in fibroblasts from 3 cases with a C9orf72 repeat extension (C9) and 1 control case (Ct). Arrows indicate Cy3-positive antisense RNA foci in the nucleus. c, d Quantification of RNA foci number and positivity upon siRNA-mediated knockdown of hnRNPA3. N = 126–234 cells from 3 biological replicates in each line. e Western blotting of cell lysates from iPSC-derived human neurons. Western blotting with antibodies to β-actin and βIII-tubulin confirmed selective and efficient knockdown of hnRNPA3. f FISH of antisense RNA foci in neurons from control cases (Ct) and C9orf72 carriers (C9). siCt : control siRNA, siA3 : hnRNPA3-targeted siRNA. g, h Knockdown of hnRNPA3 increases antisense RNA foci number and positivity in iPSC-derived neurons from C9orf72 mutation carriers. N = 72–102 cells from 3 biological replicates in each line. Data of 6 images in each line were used for analysis. NT non-treatment, siCt control siRNA, siA3 hnRNPA3-targeted siRNA, NCL nucleolin. All graphs are shown as mean ± SEM. *p < 0.05; two tailed paired t test. Scale bar 10 µm