Anti-CENPF antibody

• WB

Unknown

Western blot - Anti-CENPF antibody (AB5)

All lanes:

Western blot - Anti-CENPF antibody (ab5) at 1/1500 dilution

Lane 1:

Mitotic HeLa Lysate at 25 µg

Lane 2:

Asynchronous HeLa Lysate at 25 µg

Lane 3:

50gamma Mitotic HeLa Lysate at 25 µg

Lane 4:

50gamma Asynchronous HeLa Lysate at 25 µg

Predicted band size: 367 kDa

Observed band size: 310 kDa

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• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

ICC/IF image of ab5 stained human HeLa cells. The cells were methanol fixed (5 min) and incubated with the antibody (ab5, 1μg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iT^TM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

Interpahse and Prophase HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image emphasizes the redistribution of CENPF from the nuclear matrix during late G2 following entry into the initial stages of mitosis (see the accompanying image). Distinct punctate CENPF patterns proximally located in relation to the centromeres can be seen. In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are green and red respectively. 100x magnification.

This image is courtesy of Kirk McManus, University of British Columbia

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CENPF antibody (AB5)

ab5 staining Human normal colon tissue. Staining is localised to nuclear compartment.
Left panel : with primary antibody at 4 ug/ml. Right panel : isotype control.
Sections were stained using an automated system DAKO Autostainer Plus , at room temperature : sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

• ICC/IF

AbReview33665****

Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

Paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized A549 (human lung carcinoma cell line) cells stained for CENPF (red) using ab5 at 1/500 dilution in ICC/IF. ). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 555) at 1/500 dilution was used as the secondary antibody.

This image is courtesy of an Abreview submitted by Dr. Stuart Rulten.

• ICC/IF

Collaborator

Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

HeLa cells were labelled with anti-ACA and anti-CENPF (ab5). ab5 was used at a working dilution of 1/400. This image demonstrates the dramatic increase in fluorescence that occurs late in G2 cells (indicated by arrows). In the final panel DAPI is pseudo-coloured blue, while ACA and CENPF are coloured green and red respectively. 40x magnification.

This image is courtesy of Kirk McManus, University of British Columbia

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

Immunocytochemistry-immunofluorescence using Anti-CENPF antibody, ab5. Publication image from Stewart, G. S. et al., 2017, Mol Cell, 28238654. Legend direct from paper.

Methylation of RUVBL1 Is Required for HR, but Not ATM Signaling(A and B) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, exposed to 3Gy IR, and harvested at the time points indicated. CENPF/mitosin-positive cells were scored for RPA (A) and RAD51 (B) foci formation (mean ± SEM; n = 3).(C) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, exposed to 10Gy IR, and harvested at the time points indicated for immunoblot analysis.(D and E) HeLa-RUVBL1-WT and HeLa-RUVBL1-R205K cells were transfected with siRUVBL1, treated with increasing doses of CPT (D) or Olaparib (E), and survival was assessed by colony survival assay (mean ± SEM; n = 3). Scale bar, 10 µm. See also Figure S5.

• ICC/IF

CiteAb

Immunocytochemistry/ Immunofluorescence - Anti-CENPF antibody (AB5)

Immunocytochemistry-immunofluorescence using Anti-CENPF antibody, ab5. Publication image from Stewart, G. S. et al., 2017, Mol Cell, 28238654. Legend direct from paper.

PRMT5-Dependent Methylation of RUVBL1 at R205 Regulates 53BP1 Removal and Is Epistatic with TIP60(A) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and cell viability was assessed by colony survival. Results are expressed normalized to the respective non-treatment control (mean ± SD; n = 3).(B and C) HeLa-shCTRL or shPRMT5 cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (B) or CENPF/RPA (C) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3).(D and E) HeLa-shCTRL or RUVBL1-R205K cells were siRNA transfected, exposed to 3Gy IR, and stained for 53BP1 (D) or CENPF/RPA (E) foci 24 and 8 hr after damage, respectively (mean ± SEM; n = 3).(F) Validation of siTIP60 knockdown is shown.(G) Cells were transfected with si53BP1 and the number of mitosin-positive cells with >10 RAD51 foci was quantified (mean ± SEM; n = 3).(H) Cells were transfected with si53BP1 and the number of CENPF-positive cells with >10 RPA foci was quantified (mean ± SEM; n = 3).(I) Validation of 53BP1 siRNA SMARTpool. See also Figure S6.

• WB

CiteAb

Western blot - Anti-CENPF antibody (AB5)

Western Blotting using Anti-CENPF antibody, ab5. Publication image from Tsai, S. Y. et al., 2016, Nat Commun, 27108958. Legend direct from paper.

miRNA-COUP-TFII-CENPF-FOXM1 regulation axis contributes to the enzalutamide resistance.(a) Parental cell (LNCaP) and enzalutamide-resistant (EnzaR) clones were treated with 10 µM enzalutamide for indicated time point and cell growth was measured by MTS assay (n=3). *P<0.001 (two-way ANOVA test). (b) A representative western blot shows the expression of COUP-TFII, CENPF, FOXM1, AR and GR (glucocorticoid receptor), a positive control gene for enzalutamide-resistant clones44. (c) RT-real-time PCR data demonstrated the expressions of miR-101 and miR-27a in LNCaP (WT) and EnzaR clones (n=3). *P<0.05 (one-way ANOVA followed by Dunnett test). (d) LNCaP (WT) and EnzaR cells were used to perform migration chamber assay for 16 h (n=3). Then, invaded cells were counted and normalized to total cell number. *P<0.0001 (one-way ANOVA followed by Dunnett test) (e,f) Knockdown of COUP-TFII, CENPF or FOXM1, or overexpression of miR-101 or miR-27a in enzalutamide clone (#2) was used to perform migration chamber assay for 16 h (n=3). *P<0.0001 (one-way ANOVA followed by Dunnett test) (e) or measure cell growth by MTS assay after treating with or without 10 µM enzalutamide for 3 days (f). *P<0.0001 (one-way ANOVA followed by Dunnett test). ANOVA, analysis of variance.

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• WB

CiteAb

Western blot - Anti-CENPF antibody (AB5)

Western Blotting using Anti-CENPF antibody, ab5. Publication image from Tsai, S. Y. et al., 2016, Nat Commun, 27108958. Legend direct from paper.

miR-101-inhibited COUP-TFII leads to FOXM1 and CENPF downregulation in PCa.(a,b) Levels of CENPF and FOXM1 in LNCaP cells having overexpression of miR-101and miR-27a for 72 h (n=3) (a) and in 22RV-1 stably transfected with control, anti-miR-101 or miR-27a vectors (n=3) (b). *P<0.05 (two-sided Student's t-test) compared with the control group. (c) 22RV-1 cells treated with 50 nM miR-101 inhibitor and 25 nM siRNA against FOXM1 or CENPF were plated into invasion chamber for 16 h (n=3). Invaded cell numbers were stained and counted. *P<0.05 (two-sided Student's t-test) compared with the control group. RT-qPCR (d) and representative western blot (e) shows the levels of FOXM1, CENPF and miR-101 from PC3 cells carrying an inducible miR-101 gene in the absence and presence of doxycycline (n=3). Also shown in this figure is the rescue of FOM1 and CENPF expression when COUP-TFII is ectopically expressed for 96 h. *P<0.05 (two-sided Student's t-test) (f) miR-101, COUP-TFII, FOXM1, CENPF and FOXM1-CENPF co-regulated gene signatures were analysed in primary PCa specimens for three different expression data sets of human tumours. Yellow, high-signature scoring in prostate tumour specimens, is indicative of high manifestation of associated transcriptional patterns; blue, equals low-signature scoring. Correlation between miR-101 signature scores and scores for other signatures were tested by Pearson's correlation.

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• WB

CiteAb

Western blot - Anti-CENPF antibody (AB5)

Western Blotting using Anti-CENPF antibody, ab5. Publication image from Tsai, S. Y. et al., 2016, Nat Commun, 27108958. Legend direct from paper.

FOXM1 and CENPF are downstream targets of COUP-TFII in PCa.(a) Gene expression profile of COUP-TFII knockdown PC3 cells (GSE33182) was downloaded from GEO database and analysed using GenePattern. Genes with P<0.05, FOLD CHANGE >1.5-fold are represented as a heatmap. The gene symbol of FOXM1 and CENPF genes after knockdown of COUP-TFII are annotated in red colour. The others were FOXM1 and CENPF co-regulated genes annotated in black colour. siCtrl : knockdown scramble control; siCII : knockdown of COUP-TFII. (b) A representative western blot shows the level of COUP-TFII in PC3 cells transfected with two different siRNAs against COUP-TFII for 72 h (upper panel). Levels of FOXM1 and CENPF are quantified by RT-qPCR (lower panel) (n=3). *P<0.05 (two-sided Student's t-test) compared with the control group (siCON). (c) A cartoon shows the FOXM1 and CENPF loci. COUP-TFII-binding sites are annotated by a black rectangle (upper panel). ChIP results are shown in the PC3 cells treated with control and two different siRNAs against COUP-TFII for 72 h (lower panel) (n=3). *P<0.05 (two-sided Student's t-test) compared with control. (d) FOXM1 and CENPF promoter activities were measured in PC3 cells carrying an inducible COUP-TFII gene in the absence or the presence of doxycycline for 48 h (n=3). *P<0.05 (two-sided Student's t-test). (e) FOXM1 and CENPF promoter activities were measured in control or stably knocked-down COUP-TFII in PC3 cells for 48 h (n=3). *P<0.05 (two-sided Student's t-test). (f) PC3 cells carrying with inducible COUP-TFII-Flag gene were treated with or without doxycycline for 48 h and then transfected with individual siRNA against FOXM1 and CENPF or double knockdown of FOXM1 and CENPF for an additional 48 h. western blot (left panel) shows the level of COUP-TFII, CENPF and FOXM1 expression. Invasion assay (right panel) was performed by using those cells (n=3). *P<0.05 (two-sided Student's t-test) compared with control.

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