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Coilin is a nuclear protein that is a component of Cajal bodies, which are nuclear suborganelles involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs) and the maturation of small nucleolar RNAs (snoRNAs). Coilin serves as a marker for Cajal bodies and is essential for their formation and structural integrity.

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Lab products found in correlation

Nis elements ar 4, by Nikon (2 mentions) Fibrillarin, by Merck Group (2 mentions) Rpa194, by Santa Cruz Biotechnology (2 mentions) Cenpa, by Thermo Fisher Scientific (2 mentions) Alexa 555, by Thermo Fisher Scientific (1 mentions) Sc 35, by Abcam (1 mentions) Cf405s, by Biotium (1 mentions) Ha tag, by Roche (1 mentions) Myc tag, by Cell Signaling Technology (1 mentions) Lsm 780 nlo microscope, by Zeiss (1 mentions) Gemin2, by Santa Cruz Biotechnology (1 mentions) Flag epitope, by Merck Group (1 mentions) Fluorescence plate reader, by Tecan (1 mentions) Chemidoc image system, by Bio-Rad (1 mentions) Eclipse ti e inverted, by Nikon (1 mentions) Lsm 700, by Zeiss (1 mentions) Axio observer z1, by Zeiss (1 mentions) Zen 2009, by Zeiss (1 mentions) Flag tag (flag), by Thermo Fisher Scientific (1 mentions) Fibrillarin, by Cell Signaling Technology (1 mentions)

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Anti-Coilin (4 citations) Rabbit α-coilin (2 citations)

5 protocols using coilin

1

Multicolor immunofluorescence protocol

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Cells were transfected in 8-well glass chambers (Millipore) and fixed with 4% paraformaldehyde 24 hours later. Cells were permeabilized with 0.1% Triton-X100 and blocked with 10% donkey serum. GFP and mCherry fluoresce was detected directly. The primary antibodies used were: SC-35 (1:300; Abcam, ab11826), PML (1:50; Santa Cruz, sc-966), coilin (1:500; Santa Cruz Biotechnology, sc-32860), B23 (1:200, Santa Cruz Biotechnology, sc-56622) Myc-tag (1:500 Cell Signaling Technologies, 71D10), HA-tag (1:250; Clone 3F10, Roche, 11867423001), FK2 (1:50; Enzo Life Sciences, BML-PW8810), V5-tag (1:300, Novus Biological, NB600–379). The secondary antibodies used were Alexa 555, 647 (1:5,000; Thermo-Fisher), and CF405S (1:1000; Biotium). Samples were mounted on ProLong Gold antifade with or without DAPI and cured before imaging on a Zeiss LSM 780 NLO microscope. Images were prepared with the Fiji software.
Bouchard J.J., Otero J.H., Scott D.C., Szulc E., Martin E.W., Sabri N., Granata D., Marzahn M.R., Lindorff-Larsen K., Salvatella X., Schulman B.A, & Mittag T. (2018). Cancer mutations of the tumor suppressor SPOP disrupt the formation of active, phase-separated compartments. Molecular cell, 72(1), 19-36.e8.
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2

Quantitative Western Blot Analysis of INTS Protein Knockdown

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Western blots were performed using SDS/PAGE as described previously (33 (link)). Western blots were conducted using antibodies raised to INTS1 (Bethyl), INTS3 (Bethyl), INTS4 (Bethyl), INTS7 (Bethyl), INTS9 (Bethyl), INTS10 (Bethyl), INTS11 (Bethyl), INTS12 (Bethyl), tubulin (Abcam), Coilin (Santa Cruz), SMN (Santa Cruz), Gemin2 (Santa Cruz), GFP (Clontech) and FLAG epitope (Sigma). In Figure 3F, the degree of knockdown was determined using a Chemidoc Image System (Biorad) (Supplementary Figure S1A and B). In Figure 3G and H, the degree of RNAi-rescue was normalized to the amount of GFP expression observed in control siRNA transfected cells relative to that observed in knockdown cells (Supplementary Figure S1C and D). In the case Figure 4B, the U7-GFP reporter results were quantified using a fluorescence plate reader (Tecan) and normalized to mCherry transfection control.
Albrecht T.R., Shevtsov S.P., Wu Y., Mascibroda L.G., Peart N.J., Huang K.L., Sawyer I.A., Tong L., Dundr M, & Wagner E.J. (2018). Integrator subunit 4 is a ‘Symplekin-like’ scaffold that associates with INTS9/11 to form the Integrator cleavage module. Nucleic Acids Research, 46(8), 4241-4255.
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3

Immunofluorescence Staining of Nuclear Proteins

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Cells were fixed in 4% paraformaldehyde for 15 min and washed in phosphate-buffered saline (PBS) three times of 10-min each before permeabilized by 0.2% Triton-X 100/PBS for 10 min and additional PBS wash three times. Primary antibodies were then incubated for 1 h at room temperature. They are from the indicated sources and used at the stated dilutions: RPA194, Santa Cruz Biotechnology, catalogue#sc-48385, 1:100; PTB (S. Huang laboratory), 1:300; UBF, Santa Cruz Biotechnology, catalogue#: sc-13125, 1:50; fibrillarin, Sigma, catalogue#: ANA-N, 1:10; CENPA (Thermo Fisher #MA1-20832, 1:100; coilin, Santa Cruz Biotechnology, catalogue#: sc-32860, 1:300; Flash histone locus protein (Joseph Gall, Carnegie Institution for Science), 1:500; SMN, Santa Cruz Biotechnology, catalogue# sc-15320, 1:300, SC-35 1:200 (David Spector, Cold Spring Harbor Laboratory). After incubation with primary antibodies, the fluorescein-conjugated (Jackson ImmunoResearch) and Alexa Fluor-labeled (ThermoFisher Scientific ) secondary antibodies were incubated with cells at a dilution of 1:200 for 1 h at room temperature. Cell bearing coverslips were then mounted with Vector mounting media. Slides were visualized on a Nikon Eclipse Ti-E inverted fluorescence microscope using NIS-Elements AR 4.2 software (Nikon).
Wang C., Ma H., Baserga S.J., Pederson T, & Huang S. (2023). Nucleolar structure connects with global nuclear organization. Molecular Biology of the Cell, 34(12), ar114.
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4

Immunofluorescence Analysis of Nuclear Bodies

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0.1×106 HeLa cells were seeded in a 13 mm cover slip for 5 hours and then fixed with 4% formaldehyde (Thermo Scientific) for 20 minutes at room temperature. Suspension cells were fixed in 4% formaldehyde and then cytospun at 300 rpm for 5 min onto microscope slides. Fixed cells were permeabilised in PBST buffer (1%BSA; 0.1%Triton-X in PBS) for 30 minutes in a humidified chamber, then blocked with 5% goat serum (SIGMA) in PBST. Permeabilised cells were stained for 1 hour at room temperature with one or two of the following primary antibodies: FLAG (SIGMA; Thermo Scientific); coilin (Santa Cruz); SMN, GEMIN5 (Millipore); Fibrillarin (Cell Signalling); REIIBP [8] (link) and then for 1 hour with secondary Alexa Fluor (448, 566 or 633) antibodies. Stained cells were mounted using Vectashield media with DAPI. Confocal analyses were conducted on a Zeiss LSM700 equipped with inverted Axio Observer.Z1 and AxioCam. The lenses used were Plan-Apochromat 40×/1.3 and 63×/1.40. Immersion oil used is Immersol 518F (ZEISS). Images were acquired using ZEN 2009 (ZEISS) software and analysed using ZEN 2009 (ZEISS) or Image J softwares.
Mirabella F., Murison A., Aronson L.I., Wardell C.P., Thompson A.J., Hanrahan S.J., Fok J.H., Pawlyn C., Kaiser M.F., Walker B.A., Davies F.E, & Morgan G.J. (2014). A Novel Functional Role for MMSET in RNA Processing Based on the Link Between the REIIBP Isoform and Its Interaction with the SMN Complex. PLoS ONE, 9(6), e99493.
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5

Immunofluorescence Staining and Microscopy

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Primary antibodies were from the indicated sources and used at the stated dilutions: RPA194, Santa Cruz Biotechnology, cat #sc-48385, 1:100; PTB (S. Huang laboratory), 1:300; UBF, Santa Cruz Biotechnology, cat#: sc-13125, 1:50; fibrillarin, Sigma, cat #: ANA-N, 1:10; CENPA (Thermo Fisher #MA1-20832, 1:100; coilin, Santa Cruz Biotechnology, cat#: sc-32860, 1:300; Flash histone locus antibody protein (Gall lab), 1:500; SMN, Santa Cruz Biotechnology, cat.# sc-15320, 1:300. The fluorescein conjugated (Jackson ImmunoResearch) and Alexa Fluor-labeled (ThermoFisher) secondary antibodies were used at a dilution of 1:200. Slides were visualized on a Nikon Eclipse Ti-E inverted fluorescence microscope using NIS-Elements AR 4.2 software (Nikon).
Wang C., Ma H., Baserga S.J., Pederson T, & Huang S. (2023). Nucleolar structure connects with global nuclear organization. bioRxiv.
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