Cells were seeded on 8-well chamber slides. 48 h post-seeding, cells were pre-extracted with cold 0.5% NP-40 for 4 min on ice. Cells were then fixed with 4% PFA/PBS for 15 min and washed three times with PBS. Following washes, the slide was blocked for 1 h at room temperate with 2% BSA in PBS. Slides were then incubated with primary antibody overnight at 4ºC (anti-RNAP II CTD 8WG16 (Abcam cat: ab817) alone; anti-PCNA (Santa Cruz cat: sc-7907) alone; anti-γH2AX (Millipore cat: 05-636 JBW301, clone JBW301) alone, or anti-RNAP II CTD 8WG16 with anti-PCNA; anti-γH2AX with anti-RNAP II CTD 8WG16, or anti-γH2AX with anti-PCNA). All antibodies were used at 1:500 dilution. The next day, slides were washed three times with PBS and incubated in a pre-mixed solution of PLA probe anti-mouse minus and PLA probe anti-rabbit plus (Sigma cat: DUO92101) for 1 h at 37ºC. The Duolink In Situ detection reagents (Sigma cat: DUO92101) used to perform the PLA reaction according to the manufacturer's instructions. Slides were counterstained with DAPI and sealed with cover glass using MOWIOL. Slides were allowed to dry overnight before visualization and analysis. All immunofluorescence images were taken and analyzed using a Lecia DM4000 B fluorescence microscope using 100X magnification as indicated in figure legends.
Anti pcna
Manufactured by Merck Group
Sourced in United States
Anti-PCNA is a lab equipment product used to detect the presence and abundance of PCNA (Proliferating Cell Nuclear Antigen), a protein involved in cell proliferation. This product allows researchers to quantify PCNA levels in various biological samples.
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Lab products found in correlation
Anti cyclin d1, by Cell Signaling Technology (2 mentions)
Anti p27, by Cell Signaling Technology (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Anti flag, by Merck Group (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Anti β actin, by Merck Group (2 mentions)
Vimentin, by Novus Biologicals (2 mentions)
594 anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Anti mouse igg2a 488, by Thermo Fisher Scientific (2 mentions)
E cadherin, by Cell Signaling Technology (2 mentions)
Ecl chemiluminescence system, by Cytiva (1 mentions)
Anti usp7, by Fortis Life Sciences (1 mentions)
Anti dnmt1, by Cell Signaling Technology (1 mentions)
Anti gh2ax, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti cyclin d1, by Merck Group (1 mentions)
Anti nf200, by Merck Group (1 mentions)
30 protocols using anti pcna
1
Protein-protein Interaction Mapping via PLA
2
Immunoblotting Procedures and Antibodies
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Western blotting was performed as described [19 (link), 20 (link)]. Immunoblotting experiments were carried out according to standard procedures and visualized using the ECL chemiluminescence system (Amersham/ Pharmacia Biotech).
The following antibodies were utilized: anti-CCDC6 (Abcam), anti-USP7 (Bethyl), anti-tubulin, (SIGMA-Aldrich, Inc), anti-PCNA (Millipore), anti-DNMT1 (Cell Signaling), anti-gH2AX (#05636) was from Millipore. Secondary antibodies were from Biorad, California.
The following antibodies were utilized: anti-CCDC6 (Abcam), anti-USP7 (Bethyl), anti-tubulin, (SIGMA-Aldrich, Inc), anti-PCNA (Millipore), anti-DNMT1 (Cell Signaling), anti-gH2AX (#05636) was from Millipore. Secondary antibodies were from Biorad, California.
3
Immunofluorescence Staining of Neural Markers
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Control and treated cells were given a washing with chilled 1X PBS followed by fixation with acetone and methanol (1:1) and permeabilization with 0.3% Triton- X100 in 1X PBS. Cells were then blocked with 2% BSA and incubated with primary mouse monoclonal antibody anti-α-Tubulin (1:500), anti-NF-κB (1:500), anti-MAP-2 (1:250), anti-NF200 (1:500), anti-GAP 43 (1:250), anti-HSP70 (1:500), anti-Mortalin (1:500), anti-Bcl-xL (1:200), anti-Cyclin D1(1:250), anti-NCAM (1:250), rabbit monoclonal anti-AP-1(1:250) (all from Sigma-Aldrich) and mouse monoclonal anti-PCNA (1:250), mouse polyclonal anti-PSA-NCAM (1:250) (from Millipore, MA, USA) for 24 h in humid chamber at 4 °C. No permeabilization was carried out for PSA-NCAM immunostaining. After primary antibody incubation, three washings were given with 0.1% PBST and incubated with secondary antibody (goat anti-mouse/ rabbit IgG/ IgM Alexa Fluor 488/543) for 2 h at RT. Cells were stained with nuclear staining dye DAPI (Sigma-Aldrich) for 15 min, washed with 0.1% PBST and mounted with antifading agent Fluoromount (Sigma-Aldrich). Images were captured with Nikon AIR Confocal Laser Scanning Microscope and analyzed with NIS elements analysis software version 4.11.00 (Nikon Co., Tokyo, Japan). Each experiment was carried out in triplicate.
4
Protein expression analysis by Western blot
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Cells were harvested and lysed with RIPA lysis buffer (Beyotime Biotechnology, China). The total proteins were quantified by Bradford protein assay kit (PIERCE). The proteins were separated by SDS-PAGE and transferred into PVDF membrane. Membranes were blocked in blocking buffer (5% non-fat dry milk/0.1% Tween 20 in TBS) for 1h at room temperature, before being incubated at 4°C with the appropriate antibody in blocking buffer. The membranes were washed and incubated with the appropriated peroxidase conjugated secondary antibody. After washing, the proteins level was detected using ECL reagents (GE Healthcare). The following primary antibodies were used: anti-PCNA (Millipore, 1:250 dilution), anti-Bax (HangZhou HuaAn Biotechnology, China, 1:100 dilution), anti-Bcl-2 (Antibody Revolution, 1:250 dilution), anti-full length caspase-3 (Antibody Revolution, 1:100 dilution), and anti-dCK (Biorbyt, 1:500 dilution). Anti-rabbit (Pierce, 1:6000 dilution) or anti-mouse HRP-conjugated antibodies (Pierce, 1:3000 dilution) were used for secondary antibody reactions.
5
Identifying Proliferating Cells in Aortic Tissue
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Immunostaining of the aortic rings to identify proliferating cells with proliferating cell nuclear antigen (PCNA) was performed for confirmation of cellular proliferation [32 (link)]. Aortic ring sections (5 μm) were fixed with 4% paraformaldehyde, treated with 3.0% H2O2 for 15 minutes for endogenous peroxidase quenching, and blocked for 30 minutes in 10% normal goat serum. Sections were then washed with PBS (2x2 minutes) and incubated in 1:1000 anti-PCNA (Millipore) for 1 hour at room temperature. Upon washing, the samples were incubated with Alexa Fluor 488–conjugated anti-mouse secondary antibody for 10 minutes. The sections were then counterstained with DAPI (300 nM) for 2 minutes to display cell nuclei. The samples were quantitatively scored by a third party observer who was blinded to the study. PCNA staining was quantified as a percent of PCNA positive cells (Number of PCNA positive nuclei/Total number of nuclei).
6
Comprehensive Immunostaining Panel for Neural Cell Types
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Antibodies for immunostaining included chicken anti-GFP (1:1000 dilution, Abcam, Cat# ab13970), rat anti-GFP (1:1000, Nacalai Tesque, Cat# GF090R), anti-Sox2 (1:200, Cell Signaling Technology, Cat# 3728), anti-RFP (1:1000, MBL, Cat# PM005), anti-Fabp7 (BLBP, 1:500, Millipore, Cat# ABN14), mouse anti-S100β (1:200, Sigma-Aldrich, Cat# S2657), rabbit anti-S100β (1:500, Abcam, Cat# ab52642), anti-NICD1 (1:200, Cell Signaling Technology, Cat# 4147), anti-GFAP (1:1000, Abcam, Cat# ab4674), anti-Hey1 (1:200, Millipore, Cat# AB5714), anti-Ascl1 (1:200, BD Biosciences, Cat# 556604), anti-PCNA (1:500, Millipore, Cat# NA03), anti-Dcx (1:1000, Abcam, Cat# ab18723), anti-EGFR (1:500, Fitzgerald, Cat# 20-ES04), anti-Vcam1 (1:500, BD Biosciences, Cat# 550547), Hoechst 33342 (1:10000, Molecular Probes).
7
Comprehensive DNA Damage Response Assay
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The following antibodies were used: anti-KDM5A (D28B10-Cell Signaling Technology), anti-KDM5B (CL1147-Thermo Fisher Scientific), anti-RRM2 (2G1D5-Cell Signaling Technology), anti-CHK1 (2G1D5-Cell Signaling Technology for western blot; C9358-Sigma-Aldrich for PLA), anti-CHK1 phospho-Ser345 (133D3-Cell Signaling Technology), anti-ATR (E1S3S-Cell Signaling Technology), anti-ATR phospho-Thr1989 (GTX128145, GeneTex), anti-RPA (Subunit 9H8) (Santa Cruz Biotechnology SC56770), anti-RPA phospho-Ser33 (A300-246A-T- Bethyl), anti-PCNA (CBL407-Millipore), anti-RAD51 (SC8349-Santa Cruz Biotechnology), anti-BRCA1(SC642-Santa Cruz Biotechnology), anti-H3K4Me3 (12209-Abcam), anti-H3 (1791-Abcam), anti-GAPDH (MAB374-Millipore), anti-γH2AX (Ser139) (20E3-Cell Signaling Technology or JBW301, Millipore), anti-53BP1 (NB100-304-Novus).
8
Immunoprecipitation and Immunoblotting Assays
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Immunoprecipitation and immunoblotting experiments were performed as described before.11 (link) Briefly, the cells were first transfected with the plasmids or treated with different drugs. Then the cells were collected and washed with PBS, followed by cell lysis in the RIPA buffers.11 (link) The resultant supernatant was collected and the antibodies were added as needed. After incubation overnight at 4°C, Protein A or G Sepharose was added into the solution and incubated further for 1 hour at 4°C. The immunoprecipitates were then subject to extensive wash, then the pellets after centrifugation were collected and boiled in Sample Buffers. The following primary antibodies were used for immunoblotting: anti-MDC1, anti-GFP, anti-UBE3D, anti-PCNA, anti-KAP1, anti-HA, and anti-FLAG M2 (Sigma). Peroxidase-conjugated secondary antibodies were from Jackson Immuno Research. Blotted proteins were visualized using the ECL detection system (Amersham). Signals were detected by a LAS-4000, and analyzed using Multi Gauge (Fujifilm). Micrococcal nuclease assays were carried out as previously described.9 (link) All IP experiments were repeated for at least three times.
9
Immunohistochemical Analysis of Liver Tissue
10
Analysis of Cell Cycle Regulators
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After cell-cycle arrest, 1 × 106 cells were replated in DMEM with 10% FBS with or without PD0325901 (1 or 10 µM) or 0.1% DMSO in a 60-mm dish, and incubated for 48 h. The cells were obtained using radioimmunoprecipitation assay buffer (RIPA buffer; Thermo Fisher Scientific K.K., Kanagawa, Japan) and protease inhibitors (Roche Diagnostics, Basel, Switzerland). A BCA Protein Assay Kit (Thermo Fisher Scientific K.K.) was used to ascertain the protein quantities in the supernatant. Western blotting was performed as described previously27 (link). The following primary antibodies were incubated at 4 °C overnight: anti-PCNA (1:1000; Sigma-Aldrich), anti-p27 (1:1000; Cell Signaling Technology), anti-cyclin D1 (1:1000; Cell Signaling Technology), and anti-β-tubulin (1:1000; FUJIFILM Wako Pure Chemical). Horseradish peroxidase-conjugated secondary antibody (H goat anti-rabbit or anti-mouse IgG; 1:5000; Thermo Fisher Scientific) was incubated for 1 h at room temperature. An ImageQuant LAS 4000 mini-instrument (GE Healthcare, Chicago, IL, USA) was used to detect protein bands. WB Stripping Solution (Nacalai Tesque) was used to strip membranes of antibodies. The density of protein bands was measured using ImageJ ver. 1.53 (National Institutes of Health, Bethesda, MD, USA). Protein expression was quantified against that of β-tubulin.
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