Cenpf

Manufactured by Abcam

CENPF is a protein that is involved in the structure and function of the centromere, a key part of the chromosome responsible for chromosome segregation during cell division. It plays a role in the regulation of kinetochore assembly and function. CENPF is commonly used as a marker for cell cycle progression.

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Lab products found in correlation

Ecl detection reagent, by Beyotime (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Vimentin, by Abcam (1 mentions) E cadherin, by Abcam (1 mentions) N cadherin, by Abcam (1 mentions) β actin, by Abcam (1 mentions) Enhanced chemiluminescence detection kit, by PerkinElmer (1 mentions) Foxm1, by Cell Signaling Technology (1 mentions) Coup tfii, by R&D Systems (1 mentions) Sc 816, by Santa Cruz Biotechnology (1 mentions) E cadherin, by Cell Signaling Technology (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Dna pkcs, by Cell Signaling Technology (1 mentions) Rad50, by Cell Signaling Technology (1 mentions) Pdna pkcs s2056, by Cell Signaling Technology (1 mentions) Pkap1 s824, by Fortis Life Sciences (1 mentions) Patm s1981, by Abcam (1 mentions) Pericentrin, by Abcam (1 mentions) α tubulin b 5 1 2, by Merck Group (1 mentions) Actin 1 19, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

Anti-CENPF (4 citations) CENPF (Ab-5, (3 citations) Rabbit anti-CENPF (2 citations)

8 protocols using cenpf

Protein Expression Analysis by Western Blot

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Total proteins were extracted using 100 μl of lysis buffer containing RIPA (99 μl) and protease inhibitor cocktail (1 μl). The proteins (about 50 μg) were resolved by 10% SDS‐poly acrylamide gel electrophoresis (SDS‐PAGE), and transferred to PVDF membranes (Millipore). Then primary antibodies including Vimentin (ab8978, 1:1000), E‐cadherin (ab1416, 1:500), N‐cadherin (ab76011, 1:5000), CENPF (ab5, 1:500) and β‐actin (ab6276, 1:1000) (Abcam) were applied to probe with the membranes all night at 4°C. After the secondary incubation at 37°C for 2 h, the ECL detection reagent (Beyotime) was employed for the detection of protein bands.

Liu Y., Liu Y., Luo J., Zhao W., Hu C, & Chen G. (2022). Hsa_circ_0002082 up‐regulates Centromere Protein F via abolishing miR‐508‐3p to promote breast cancer progression. Journal of Clinical Laboratory Analysis, 36(11), e24697.

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Comprehensive Protein Expression Analysis

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Total cell lysates were collected by RIPA buffer containing commercial protease inhibitors. 30 μg of protein was separated by SDS-polyacrylamide gel electrophoresis, and transferred to a polyvinylidene difluoride membrane. The membrane was blocked in 5% nonfat milk at room temperature for 1 h, followed by incubation with primary antibody prepared in 1 × PBST (PBS with 0.25% Tween 20) (COUP-TFII, 1:1000, PP-H7147-00, R&D systems; CENPF, 1:2000, Ab5, Abcam; FOXM1, 1:2000, #5436, Cell Signaling Technology; E-cadherin, 1:2000, #3195, Cell Signaling Technology; Vimentin, 1:2000, #5741 Cell Signaling Technology; GR, 1:2000, #12041, Cell Signaling Technology and AR, 1:2000, sc-816, Santa Cruz Biotechnology Inc.) at 4 °C overnight. Signals were developed using an enhanced chemiluminescence detection kit (PerkinElmer). For IHC staining, AR (1:1,000) antibody was purchased from Santa Cruz Biotechnology Inc (sc-816) and GFP (1:1,000) antibody was purchased from ThermoFisher (A11122). Uncropped scans of blots are shown in Supplementary Figs 15–17.

Lin S.C., Kao C.Y., Lee H.J., Creighton C.J., Ittmann M.M., Tsai S.J., Tsai S.Y, & Tsai M.J. (2016). Dysregulation of miRNAs-COUP-TFII-FOXM1-CENPF axis contributes to the metastasis of prostate cancer. Nature Communications, 7, 11418.

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Antibody-based Analysis of DNA Damage Repair

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Antibodies against the following proteins were used for immunofluorescence (IF) and Western blotting (WB): γH2AX (05–636 JBW301; Upstate Biotechnology), CENPF (ab5; Abcam), RPA (NA-18, Ab-2; Calbiochem for IF, LS-C38952; LifeSpan BioSciences for WB), pRPA S4/8 (A300-245A; Bethyl Laboratories), p DNA-PKcs S2056 (ab18192), DNA-PKcs (12311; Cell Signaling), Ku80 (2180 (C48E7); Cell Signaling), pKAP-1 S824 (A300-767A; Bethyl Laboratories), KAP-1 (ab3831; Abcam), MRE11 (12D7, GTX70212; GeneTex), NBS1 (PC269, Ab-1; Oncogene), RAD50 (3427; Cell Signaling), pATM S1981 (2152–1; Epitomics), ATM (GTX70103 2C1; GeneTex), CtIP (A300-488A; Bethyl Laboratories), and β-tubulin (ab21058; Abcam). Ku70, XLF, XRCC4, and Ligase IV antibodies were kind gifts from Dr. Penny Jeggo. Immunoblotting was performed as described previously [17 (link)].

Nakajima N.I., Hagiwara Y., Oike T., Okayasu R., Murakami T., Nakano T, & Shibata A. (2015). Pre-Exposure to Ionizing Radiation Stimulates DNA Double Strand Break End Resection, Promoting the Use of Homologous Recombination Repair. PLoS ONE, 10(3), e0122582.

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Comprehensive Mitotic Spindle Protein Analysis

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The following antibodies were commercially purchased and used at the indicated dilutions for immunofluorescence (IF) and immunoblotting (IB); Actin (I-19, Santa Cruz Biotechnology, IB; 1/2000), α-tubulin (B-5-1-2, Sigma, IF; 1/1000), BubR1 (Bethyl Laboratories, IF; 1/500), CENP-E (Sigma, IF; 1/500, IB; 1/2000), CENP-F (Abcam, IF; 1/1000), CLIP170 (Santa Cruz Biotechnology, IF; 1/500), dynein intermediate chain (DIC, Sigma, IF; 1/500), Hec1 (9G3, Abcam, IF; 1/1000, IB; 1/2000), Kid (Cytoskeleton Inc., IB: 1/1000), KNL1 (Novus Biologicals, IF; 1/500), Mad2 (Novus Biologicals, IF; 1/500), Nde1 (Protein Tech Group, IF; 1/500), Ska1 (Abcam, IF; 1/500), Spindly (Bethyl Laboratories, IF; 1/500, IB; 1/2000), Zwint-1 (Bethyl Laboratories, IF; 1/500), Zw10 (Cosmo Bio, IF; 1/200, IB; 1/2000), pericentrin (Abcam, IF; 1/500).

Itoh G., Ikeda M., Iemura K., Amin M.A., Kuriyama S., Tanaka M., Mizuno N., Osakada H., Haraguchi T, & Tanaka K. (2018). Lateral attachment of kinetochores to microtubules is enriched in prometaphase rosette and facilitates chromosome alignment and bi-orientation establishment. Scientific Reports, 8, 3888.

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Western Blot and Immunoprecipitation Assay

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Whole protein extracts were obtained by lysing cells in TNN buffer (50mM Tris (pH 7.5), 120mM NaCl, 5mM EDTA, 0.5% NP40, 10mM Na4P₂O₇, 2mM Na₃VO₄, 100mM NaF, 1mM PMSF, 1mM DTT, 10mM β-glycerophosphate, protease inhibitor cocktail (Sigma)). Protein lysates or purified protein were separated by SDS-PAGE, transferred to a PVDF-membrane and detected by immunoblotting with the first and secondary antibodies: β-actin (Santa Cruz Biotechnology, sc-47778) 1:5000, B-MYB (clone LX015.1, [42 (link)] 1:5, anti-HA.11 (Covance, MMA-101P) 1:1000, anti-FLAG M2 (Sigma, F3165) 1:5000, anti-His (Sigma, H1029) 1:2000, Vinculin (Sigma, V9131) 1:10000, TOP2A (Santa Cruz Biotechnology, sc-365916) 1:1000, CDC20 (Santa Cruz Biotechnology, sc-5296) 1:500, YAP (Santa Cruz Biotechnology, sc-10199) 1:1000, p-YAP(S127A) (Cell Signaling Technology, 4911) 1:1000, LIN9 (Bethyl, A300-BL2981), NUSAP1 (Geert Carmeliet) 1:1000, CENPF (Abcam, ab-5) 1:1000, anti-mouse-HRP (GE healthcare, NXA931) 1:5000 and HRP Protein A (BD Biosciences, 610438) 1:5000. For immunoprecipitation of FLAG-tagged proteins, protein G dynabeads (Thermo Fisher Scientific) were first coupled with 1 μg FLAG-antibody (Sigma, F3165) and then immunoprecipitated with 1mg whole cell lysate. After five times of washing with TNN, proteins were separated by SDS-PAGE and detected by immunoblotting using the desired antibodies.

Gründl M., Walz S., Hauf L., Schwab M., Werner K.M., Spahr S., Schulte C., Maric H.M., Ade C.P, & Gaubatz S. (2020). Interaction of YAP with the Myb-MuvB (MMB) complex defines a transcriptional program to promote the proliferation of cardiomyocytes. PLoS Genetics, 16(5), e1008818.

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Western Blot Analysis of Protein Expression

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Total protein extracted from collected specimens (0.5 g per sample) was cut into small sections and homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors (Beyotime). After centrifugation at 13,000 × g, at 4°C for 20 min, the supernatant was recovered.
After separation by 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), proteins were electroblotted onto nitrocellulose membranes (Millipore) and subjected to western blot analysis, then incubated with primary antibodies, CENPF (rabbit polyclonal, 1:1,000 dilution, ab5), FOXM1 (rabbit polyclonal, 1:1,000dilution, ab226928) (both from Abcam) and GAPDH (rabbit monoclonal, 1:1,000dilution, no. 5174; Cell Signaling Technology), and membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (goat anti rabbit, 1:1,000 dilution, A0208; Beyotime). The immunoreactive signal was detected by the enhanced chemiluminescence kit (Millipore). Quantification of band intensity was analyzed with ImageJ software (http://rsb.info.nih.gov/ij/), and normalized to the intensity of GAPDH.

Li R., Wang X., Zhao X., Zhang X., Chen H., Ma Y, & Liu Y. (2019). Centromere protein F and Forkhead box M1 correlation with prognosis of non-small cell lung cancer. Oncology Letters, 19(2), 1368-1374.

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Antibody-Based Imaging and Immunoblotting

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The primary antibodies used were as follows: γH2AX (Upstate Technology; 05-636) at 1:800 for IF, RPA (Merck Millipore; LS-C38952) at 1:100 for IF, RAD51 (Santa Cruz Biotechnology; SC-8349) at 1:200 and CENP-F (Abcam; ab108483) for IF, INO80 (Abcam; ab118787, and Bethyl; A303-371A) at 1:2,000 for WB, H2A.Z (Cell Signaling Technology; 2718S) at 1:1,000 for WB, ANP32E (Sigma-Aldrich; SAB2100124) at 1:1,000 for WB, KAP1 (Abcam; ab22553) at 1:1,000 for WB and KU80 (Abcam; ab33242) at 1:1,000 for WB.
The secondary antibodies used were as follows: FITC (Sigma-Aldrich; F0257) at 1:100 for IF, Cy3 (Sigma-Aldrich; C2306) at 1:200 for IF, AlexaFluor 488 (Invitrogen; A21206) at 1:400 for IF, Goat Anti-Rabbit Immunoglobulin/HRP (Dako; P0449) at 1:2,000 for WB, Rabbit Anti-Mouse Immunoglobulins/HRP (Dako; P044801-2) at 1:2,000 for WB.

Alatwi H.E, & Downs J.A. (2015). Removal of H2A.Z by INO80 promotes homologous recombination. EMBO Reports, 16(8), 986-994.

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Immunofluorescence Staining of DNA Damage Markers

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Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton-X 100. To prevent non-specific antibody binding, slides were blocked with 1% BSA. Cells were incubated with primary antibody at room temperature for one hour (phospho-histone H2A.X (1:10000, Millipore #05–636); RAD51(1:1000, Abcam); CENPF (1:500, Abcam #5) diluted in 1% BSA. After incubation with secondary antibody (Molecular Probes, Invitrogen, UK) the nuclei were stained 100 ng/µL DAPI. Coverslips were mounted with Mowiol 4–88 (Sigma-Aldrich, UK). Cells were imaged with a Leica DMI6000 inverted epifluorescence microscope with 100x lens and Photometrics Prime 95B sCMOS camera or a Leica SPE single channel confocal laser scanning microscope with 40x lens and Leica DFC365FX monochrome digital camera.

Parker C., Chambers A.C., Flanagan D.J., Ho J.W., Collard T.J., Ngo G., Baird D.M., Timms P., Morgan R.G., Sansom O.J, & Williams A.C. (2022). BCL-3 loss sensitises colorectal cancer cells to DNA damage by targeting homologous recombination. DNA Repair, 115, 103331.

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