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Ncl 4e2

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NCL (4E2) is a mouse monoclonal antibody that recognizes the nuclear protein nucleolin. Nucleolin is a multifunctional protein involved in various cellular processes, including ribosome biogenesis, chromatin remodeling, and regulation of gene expression. The NCL (4E2) antibody can be used for the detection and study of nucleolin in different experimental applications.

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Lab products found in correlation

Axiocam hrc ccd camera, by Zeiss (3 mentions) Npm fc 61991, by Thermo Fisher Scientific (3 mentions) Axioplan 2 fluorescence microscope, by Zeiss (3 mentions) Axiovision 4, by Zeiss (2 mentions) Orca flash4.0 v2 scmos camera, by Hamamatsu Photonics (2 mentions) Alexa 488, by Thermo Fisher Scientific (2 mentions) Polr1b rpa135 4h6, by Santa Cruz Biotechnology (2 mentions) Polr1a rpa194 c 1, by Santa Cruz Biotechnology (2 mentions) Alexa 594 conjugated anti mouse and anti rabbit antibodies, by Thermo Fisher Scientific (2 mentions) Dm6000b wide field fluorescence microscope, by Leica (2 mentions) Rpa194 c 1, by Santa Cruz Biotechnology (2 mentions) Ec plan neofluar objective, by Zeiss (1 mentions) Phospho atm, by Cell Signaling Technology (1 mentions) Phospho dna pkcs, by Abcam (1 mentions) Phospho kap1, by Fortis Life Sciences (1 mentions) γh2ax, by Merck Group (1 mentions) Hrp conjugated secondary antibody, by Agilent Technologies (1 mentions) Hrp conjugated streptavidin, by Agilent Technologies (1 mentions) Enhanced chemi luminescence (ecl), by Cytiva (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)

7 protocols using ncl 4e2

1

Immunostaining Protocol for Cellular Imaging

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Immunostaining was performed essentially as in ref. (9 (link)). Cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized with 0.5% NP-40 and blocked in 3% BSA. The following primary antibodies were used: UBF (H-300) and RPA194 (C-1) (Santa Cruz Biotechnology), NCL (4E2, Abcam), NPM (FC-61991, Invitrogen), FBL (ab582, Abcam), γH2AX (Upstate), KAP-1 (BD Transduction Laboratories) and p53 (7F5, Cell Signaling Technologies). Secondary Alexa488 and Alexa594-conjugated anti-mouse and anti-rabbit antibodies were from Invitrogen. DNA was stained with DAPI. Images were captured using Axioplan2 fluorescence microscope (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar objectives (Zeiss). Image analysis was conducted using FrIDA designed for the analysis of RGB color image datasets as in ref. (9 (link)). Hue saturation and brightness ranges for green and red fluorescence channel and DNA (blue) were defined for each image set. Image intensities were determined as the fraction of positive cells divided total nuclear area as defined by DNA staining. An average of 100 cells was quantified from two fields for each sample.
Peltonen K., Colis L., Liu H., Jäämaa S., Zhang Z., Hällström T.A., Moore H.M., Sirajuddin P, & Laiho M. (2014). Small Molecule BMH-compounds that Inhibit RNA Polymerase I and Cause Nucleolar Stress. Molecular cancer therapeutics, 13(11), 2537-2546.
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2

Immunofluorescence Staining of Nucleolar Proteins

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For all immunofluorescence procedures, we followed our earlier protocols (Peltonen et al., 2014a (link)). Cells grown on coverslips were fixed in 3.5% paraformaldehyde or 100% methanol, permeabilized with 0.5% NP-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, and 50 mM NaF), and blocked in 3% BSA. The following primary antibodies were used: POLR1B/RPA135 (4H6; Santa Cruz Biotechnology), POLR1A/RPA194 (C-1; Santa Cruz Biotechnology), NPM (FC-61991; Invitrogen), NCL (4E2; Abcam), and fibrillarin (ab5821; Abcam). Secondary antibodies used were Alexa 488 and Alexa 594-conjugated anti-mouse and anti-rabbit antibodies (Invitrogen). DNA was stained with Hoechst 33342. Images were captured using DM6000B wide-field fluorescence microscope (Leica). The microscope was equipped with a Hamamatsu Orca-Flash 4.0 V2 sCMOS camera and LAS X software by using 40×/1.25–0.75 HCX PL APO CS oil and 63×/1.40–0.60 HCX PL APO Lbd.bl. oil objectives. Quantitative image analysis of nucleolar protein expression was as described in Peltonen et al. (2014a) (link) and was conducted on at least 200 cells per sample on three to five fields.
Wei T., Najmi S.M., Liu H., Peltonen K., Kucerova A., Schneider D.A, & Laiho M. (2018). Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint. Cell reports, 23(2), 404-414.
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3

Immunostaining and Image Analysis Protocol

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Immunostaining was performed essentially as in ref. [14 (link)] and ref. [30 (link)]. Cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized with 0.5% NP-40 and blocked in 3% BSA.The following primary antibodies were used: UBF (H-300, Santa Cruz Biotechnology), NCL (4E2, Abcam), RPA194 (C-1, Santa Cruz Biotechnology), phospho-ATM (Cell Signaling Technology), γH2AX (Millipore), phospho-KAP1 (Bethyl Laboratories), phospho-DNA-PKcs (Abcam). Secondary Alexa488 and Alexa594-cojugated anti-mouse and anti-rabbit antibodies were from Invitrogen. DNA was stained with DAPI. Images were captured using Axioplan2 fluorescence microscope (Zeiss) equipped with AxioCam HRc CCD-camera and AxioVision 4.5 software using EC Plan-Neofluar 20x/0.5 and 40x/0.75 objectives (Zeiss). Image analysis was conducted using FrIDA designed for the analysis of RGB color image datasets as in ref. [14 (link)] and ref. [25 (link)]. Hue saturation and brightness ranges for green and red fluorescence channel and DNA (blue) were defined for each image set. Image intensities were determined as the fraction of positive cells divided total nuclear area as defined by DNA staining. An average of 100 cells was quantified from two fields for each sample.
Colis L., Peltonen K., Sirajuddin P., Liu H., Sanders S., Ernst G., Barrow J.C, & Laiho M. (2014). DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response. Oncotarget, 5(12), 4361-4369.
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4

Western Blot Analysis of Cellular Proteins

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Lysis of cells was conducted in 0.5%
NP-40 buffer (25 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM
NaF, 100 μM Na3VO4, 100 KIU/mL aprotinin,
10 μg/mL leupeptin). Proteins were separated on SDS–PAGE
gel and blotted as in ref (14 (link)). The following antibodies were used: NCL (4E2, Abcam),
RPA194 (C-1, Santa Cruz Biotechnology), GAPDH (Europa Bioproducts).
HRP-conjugated secondary antibodies were from DAKO.
Colis L., Ernst G., Sanders S., Liu H., Sirajuddin P., Peltonen K., DePasquale M., Barrow J.C, & Laiho M. (2014). Design, Synthesis, and Structure–Activity Relationships of Pyridoquinazolinecarboxamides as RNA Polymerase I Inhibitors. Journal of Medicinal Chemistry, 57(11), 4950-4961.
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5

Western Blot Analysis of Cellular Proteins

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Cells were lysed in 0.5% NP-40 buffer (25 mM Tris-HCl, pH 8.0, 120 mM NaCl, 0.5% NP-40, 4 mM NaF, 100 μM Na3VO4, 100 KIU/ml aprotinin, 10 μg/ml leupeptin) or RIPA lysis buffer. Proteins were separated on SDS-PAGE, blotted, probed for respective proteins and detected using ECL (Amersham). The primary antibodies used for detection were NCL (4E2; Abcam), RPA194 (C-1 Santa Cruz Biotechnology). HRP-conjugated secondary antibodies and were from DAKO or Santa Cruz Biotechnology, HRP-conjugated streptavidin was from DAKO.
Colis L., Peltonen K., Sirajuddin P., Liu H., Sanders S., Ernst G., Barrow J.C, & Laiho M. (2014). DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response. Oncotarget, 5(12), 4361-4369.
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6

Immunofluorescence Staining of Nucleolar Proteins

Cited 1 time
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For all immunofluorescence procedures, we followed our earlier protocols (Peltonen et al., 2014a (link)). Cells grown on coverslips were fixed in 3.5% paraformaldehyde or 100% methanol, permeabilized with 0.5% NP-40 lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, and 50 mM NaF), and blocked in 3% BSA. The following primary antibodies were used: POLR1B/RPA135 (4H6; Santa Cruz Biotechnology), POLR1A/RPA194 (C-1; Santa Cruz Biotechnology), NPM (FC-61991; Invitrogen), NCL (4E2; Abcam), and fibrillarin (ab5821; Abcam). Secondary antibodies used were Alexa 488 and Alexa 594-conjugated anti-mouse and anti-rabbit antibodies (Invitrogen). DNA was stained with Hoechst 33342. Images were captured using DM6000B wide-field fluorescence microscope (Leica). The microscope was equipped with a Hamamatsu Orca-Flash 4.0 V2 sCMOS camera and LAS X software by using 40×/1.25–0.75 HCX PL APO CS oil and 63×/1.40–0.60 HCX PL APO Lbd.bl. oil objectives. Quantitative image analysis of nucleolar protein expression was as described in Peltonen et al. (2014a) (link) and was conducted on at least 200 cells per sample on three to five fields.
Wei T., Najmi S.M., Liu H., Peltonen K., Kucerova A., Schneider D.A, & Laiho M. (2018). Small-Molecule Targeting of RNA Polymerase I Activates a Conserved Transcription Elongation Checkpoint. Cell reports, 23(2), 404-414.
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7

Immunofluorescence Staining of U2OS Cells

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U2OS
cells grown on coverslips were fixed in 3.5% paraformaldehyde, permeabilized
with 0.5% NP-40, and blocked with 3% BSA as described in ref (13 (link)). Cells were stained for
RPA194 (C-1, Santa Cruz Biotechnology) and NCL (4E2, Abcam). Alexa
488 and Alexa 594 conjugated anti-mouse or anti-rabbit antibodies
were from Invitrogen. DNA was counterstained with DAPI (Invitrogen).
Images were captured using Axioplan2 fluorescence microscope (Zeiss)
equipped with AxioCam HRc CCD camera and AxioVision 4.5 software using
EC Plan-Neofluar 20×/0.75 objective (Zeiss).
Colis L., Ernst G., Sanders S., Liu H., Sirajuddin P., Peltonen K., DePasquale M., Barrow J.C, & Laiho M. (2014). Design, Synthesis, and Structure–Activity Relationships of Pyridoquinazolinecarboxamides as RNA Polymerase I Inhibitors. Journal of Medicinal Chemistry, 57(11), 4950-4961.
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