Anti-Pericentrin antibody - Centrosome Marker

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

ab4448 staining Pericentrin in NIH3T3 cells. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab4448 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Also suitable in cells fixed with 100% methanol (5 min).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

IF staining of pericentrin in MCF7 (Human breast adenocarcinoma cell line) cells.

The top panel is an interphase cell showing centrosome staining.

The bottom panel shows a mitotic cell with spindle pole staining.

ab4448 was used at 1/500, but also works at higher dilutions (1/1000-1/2000).

Top panel - 630X magnification; Bottom panel -1000X magnification.

The secondary antibody was Alexa-Fluor^®488 anti-rabbit.

This image is courtesy of Gordon Chan, University of Alberta

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

A representative panel of indirect immunofluorescence microscopic images shows normal (regular, n ≤ 2) and aberrant centrosome numbers (n > 2) in interphase cells.

Centrosomes were stained using anti-pericentrin antibody ab4448 (magenta), nuclear DNA is shown in blue (DAPI).

Statistical methods : Kruskal-Wallis test. Mann-Whitney U tests followed by Bonferroni-Holm p-value correction were made as post-hoc tests in order to compare the MDS and sAML patients with the control group.

Ruppenthal et al PLoS One. 2018 Jan 25;13(1):e0191734. doi: 10.1371/journal.pone.0191734. eCollection 2018. Fig 2. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

ab4448 staining Pericentrin in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab4448 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

NIH/3T3 (Mouse embryo fibroblast cell line) cells were fixed in 100% methanol for 6 minutes at -20°C, washed 3 times in PBS then incubated with ab4448 (1/2000) for 1 hour at room temperature.

The panel of images shows the nuclei stained with DAPI (blue), ab4448 staining is shown in green. 100x magnification.

This image is courtesy of Roberto Giambruno, Marilena Ciciarello and Patrizia Lavia

• WB

CiteAb

Western blot - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

Western Blotting using Anti-Pericentrin antibody - Centrosome Marker, ab4448. Publication image from Holdgaard, S. G. et al., 2019, Nat Commun, 31519908. Legend direct from paper.

The CS are autophagy substrates. a MCF7 cell extracts of stable CRISPR/Cas9 non-targeting control (CTRL), ATG5- or ATG7-transfected partial knock-out pools immunoblotted for CS and centrosome proteins. Vinculin is used as loading control. b Densitometric quantification of CS and centrosome protein levels relative to vinculin, represented in (a). Columns represent the mean ± SD, n = 3, ns P > 0.05 *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Unpaired Student’s t-test, two-tailed. c Stable CRISPR/Cas9 non-targeting control (CTRL), ATG5- or ATG7 partial knock-out MCF7 pools immunoblotted for CS proteins. Vinculin is used as loading control. d Densitometric quantification of CS protein levels relative to vinculin, represented in (c). Columns represent the mean ± SD, n = 3, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Unpaired Student’s t-test, two-tailed. e–g Immunoblot of U2OS (e), MCF7 (f) or HEK293 (g) cells treated with Baf for the indicated times and immunoblotted for PCM1, CEP131, and vinculin as loading control. h Densitometric quantification of PCM1 and CEP131 immunoblots represented in (e–g). Columns represent the mean ± SD, n = 4, ns P > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Unpaired Student’s t-test, two-tailed. i Colocalization of LC3B with PCM1 or CEP131 in U2OS cells after 6 h of Baf treatment. Arrows indicate examples of colocalization. j Colocalization between SSX2IP, PCM1, and mCherry-LC3B in U2OS cells treated for 6 h with 200 nM Baf. Arrows indicate examples of colocalization. Scale bars, 10 µm. Source data are provided as a Source Data file

false

• WB

CiteAb

Western blot - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

Western Blotting using Anti-Pericentrin antibody - Centrosome Marker, ab4448. Publication image from Guttmann-Raviv, N. et al., 2014, Nucleic Acids Res, 24728997. Legend direct from paper.

KDM4C, but not KDM4B, depletion increases chromosomal segregation errors during mitosis. (A)–(D) Representative images showing normal and defective mitotic U2OS cells depleted of KDM4C. Cells were subjected to immunofluorescence analysis using Pericentrin (green) andα-tubulin antibodies (red). DNA is stained with DAPI (blue). (A) Normal and abnormal metaphase with misaligned chromosome (indicated by white arrow). (B) Normal and abnormal anaphase with either lagging chromosomes or anaphase bridge (indicated by white arrows). (C) Abnormal telophase with either lagging chromosomes or telophase bridge (indicated by white arrows). (D) Multipolar metaphase. (E) KDM4B and KDM4C knockdown by western blotting. U2OS cells were transfected with either control or different sequences of KDM4B and KDM4C Stealth siRNA (Invitrogen). Protein extracts were prepared 72 h after transfection and immunoblotted with KDM4B and KDM4C antibody. β-Actin is used as a loading control. (F) A histogram showing the percentage of metaphases with misaligned chromosomes 72 h after transfection with control and different KDM4B-C siRNA sequences. KDM4C, but not KDM4B, depletion increases the frequency of metaphase cells with misaligned chromosomes. n, number of metaphase cells counted. Error bars represent standard deviation from two independent experiments. (G) KDM4C, but not KDM4B, depletion increases the frequency of anaphase–telophase cells with either lagging chromosomes or anaphase–telophase bridges. As in (F), except that the histogram shows the percentage of defective anaphase–telophase cells.

false

• WB

CiteAb

Western blot - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

Western Blotting using Anti-Pericentrin antibody - Centrosome Marker, ab4448. Publication image from Clift, D. et al., 2017, Cell, 29153837. Legend direct from paper.

Time Frame for Protein Depletion by Trim-Away, Related to Figure 5(A–F) NIH 3T3-mCherry-TRIM21 cells were taken up into the Neon Pipette Tip and either electroporated or not (mock). Percentage of dead cells was determined using the trypan blue exclusion assay (A). For long-term analysis of cellular behavior cells were imaged every 15 min for 70 h following electroporation (B-F). For the growth curves cell density was normalized to the initial density once cells had adhered (D). For adherence analysis the percentage of spread cells was quantified in each frame (B and C). Doubling times (E and F) were calculated taking the 5 h time point as reference, because cells were fully adhered then. Data from three independent experiments. Error bars show s.d. P values were calculated with Student’s t test. See also Movie S4.(G and H) HEK293T-mCherry-TRIM21 cells were electroporated with either control IgG, anti-ERK1 or anti-IKKα antibodies and whole cell lysates harvested at the indicated times after electroporation for immunoblotting.(I–M) NIH 3T3-mCherry-TRIM21 cells were electroporated with control IgG or anti-Pericentrin antibody (BD611815) and 3 hours later analyzed for Pericentrin (I and K) or Cdk5rap2 (J and L) localization or cell lysates were immunoblotted for the indicated proteins (M). Merge also shows DNA stained with Hoechst. Number of cells in brackets. Scale bars, 5 µm. Data from two independent experiments. P values were calculated with Fisher’s exact (K) or Student’s t test (L).

false

• WB

CiteAb

Western blot - Anti-Pericentrin antibody - Centrosome Marker (AB4448)

Western Blotting using Anti-Pericentrin antibody - Centrosome Marker, ab4448. Publication image from Clift, D. et al., 2017, Cell, 29153837. Legend direct from paper.

Trim-Away of Pericentrin by Antibody Electroporation(A–C) NIH 3T3 and HEK293T cells were electroporated with Alexa Fluor 488-labeled IgG and analyzed 3 hr later by flow cytometry (A and B) or fixed 16 hr later and analyzed by microscopy (C). At least 2,000 cells were counted for each condition. Percentages correspond to IgG-positive cells falling within the gate drawn. Scale bars, 10 µm.(D–H) NIH 3T3 and NIH 3T3-mCherry-TRIM21 cell lines were electroporated with control IgG or anti-pericentrin antibodies and analyzed 16 hr later for pericentrin (D and E) and Cdk5rap2 localization (G and H). Number of cells in brackets. Scale bars, 5 µm. DNA stained with Hoechst.(F) Cell lysates were immunoblotted for the indicated proteins. Asterisks show non-specific bands not degraded by Trim-Away.Data from two independent experiments (E and H). P values were calculated with Fisher’s exact (E) or Student’s t test (H).See also Figure S4, Figure S5, Figure S6, Figure S7.

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