The immunofluorescent staining was performed at Molecular Cytology Core Facility of XXX using Discovery XT processor (Ventana Medical Systems, Tuscon, AZ). Antigen retrieval was performed with CC1 buffer (Ventana Medical Systems). Sections were blocked for 30 minutes with Background Buster solution (Innovex, Richmond CA). Anti-phospho- H2A.X (Millipore, clone JBW301 0.03ug/mL) antibody was applied and sections were incubated for 5 hours, followed by 60 minutes incubation with biotinylated horse anti-mouse IgG (Vector Labs, Burlington CA) at 1:200 dilution. Detection was performed with Streptavidin-HRP D (Ventana Medical Systems), followed by incubation with Tyramide Alexa Fluor 568 (Invitrogen, Carlsbad CA) prepared according to manufacturer instruction with predetermined dilutions. Slides were counterstained with DAPI (5 ug/mL Sigma Aldrich, St Louis, MO) for 10 min and coverslipped with Mowiol.
Anti phospho h2ax
Manufactured by Merck Group
Sourced in United States
Anti-phospho-H2AX is a laboratory reagent used to detect DNA double-strand breaks. It specifically binds to the phosphorylated form of the histone H2AX, which is a marker for the presence of DNA damage.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Anti p chk1, by Cell Signaling Technology (2 mentions)
Anti p chk2, by Cell Signaling Technology (2 mentions)
Anti p atm, by Cell Signaling Technology (2 mentions)
Anti lig3, by Fortis Life Sciences (2 mentions)
Anti ku70, by Santa Cruz Biotechnology (2 mentions)
Anti timeless, by Fortis Life Sciences (2 mentions)
Anti parp1, by Thermo Fisher Scientific (2 mentions)
Anti hltf, by Fortis Life Sciences (2 mentions)
Anti xrcc1, by Cell Signaling Technology (2 mentions)
Anti dna pk, by Santa Cruz Biotechnology (2 mentions)
Anti dna pk, by Cell Signaling Technology (2 mentions)
Anti tipin, by Fortis Life Sciences (2 mentions)
Anti rpa1, by Santa Cruz Biotechnology (2 mentions)
Anti rpa2, by Merck Group (2 mentions)
Anti ssrp1, by Abcam (2 mentions)
Anti spt16, by Cell Signaling Technology (2 mentions)
Anti skp1, by Santa Cruz Biotechnology (2 mentions)
Anti parp1, by Cell Signaling Technology (2 mentions)
Anti flag m2, by Merck Group (2 mentions)
9 protocols using anti phospho h2ax
1
Immunofluorescent Staining of Phospho-H2A.X
2
PARP Inhibition Protein Analysis
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Cells were plated in 6 wells at a density of 2 × 105 cells per well. After PARP inhibition, cells were washed twice with PBS and resuspended in 200 μl of TR3 Lysis Buffer (3% SDS, 10% Glycerol, 10mM Na2HPO4 anhidro). Then cells were sonicated and 20 μl of 50% β-mercaptoethanol - 50% Bromofenol blue were added. The protein concentration was determined using the Lowry assay. Proteins were resolved on SDS-polyacrylamide gels and transferred onto PVDF Membrane (Biorad). The blot was blocked with 5% milk powder in PBS1X with 0.1% Tween-20 for 60 minutes and incubated overnight with 1% milk powder in PBS1X with 0.1% Tween-20 with the following antibodies: anti-PARP-1(C2-10 mouse, ALEXIS, LA), anti-PTEN (Santa Cruz Biotechnology sc-7974), anti-BRCA1 (Santa Cruz Biotechnology sc-642), anti-RAD51 (Santa Cruz Biotechnology (H-92) sc-8349), anti-phosphoERK (Santa Cruz Biotechnology sc-7383), anti-ERK (Invitrogen. Carlsbad, CA 61-7400), anti-phospho H2AX (Millipore 05-636) and anti-BUBR1 (BD Bioscience. Erembodegem, Belgium). α-Tubulin (Sigma, St Louis MO) and β-Actin (Sigma, St Louis MO) were used as loading control. Bands were visualized with ECL, ECL-PLUS and ECL PRIME (Amersham Biosciences) and the pictures were taken with the imaging system ChemiDoc XRS System (BIO-RAD) and medical X-ray films (AGFA).
3
Antibodies for DNA Damage Response Analysis
4
Immunofluorescent Analysis of DNA Damage
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HSPCs were fixed with 4% paraformaldehyde and treated with anti-phospho-H2AX (Millipore, MA, USA) or anti-53BP1 (Santa Cruz Biotechnology, CA, USA), which was diluted in blocking buffer (1% BSA/0.1% Triton X-100). Thereafter, either anti-mouse-PE or anti-rabbit-FITC (BD Bioscience) was added as a secondary antibody (1:500). The nuclei were stained with DAPI from Sigma (St. Louis, MO, USA).
5
Antibodies for DNA Damage Response Analysis
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The following antibodies were used: anti-TIMELESS (Bethyl Laboratories, A300-961A and A300-960A, the latter used for IP), anti-PARP1 (Invitrogen, 436400), anti-PARP1 (Cell Signaling Technology, 9542 and 46D11, the latter used for IF), anti-FLAG M2 (Sigma, F3165), anti-p-Chk1 (Cell Signaling, 2344), anti-p-Chk2 (Cell Signaling, 2661), anti-p-ATM (Cell Signaling, 4526), anti-LIG3 (Bethyl, A301-637A), anti-HLTF (Bethyl, A300-229A), anti-KU70 (Santa Cruz Biotechnology, sc-9033), anti-KU80 (Neomarkers, MS-285-P1; Cell Signaling, 2180), anti-XRCC1 (Cell Signaling, 2735), anti-DNA-PK (Santa Cruz, sc-5282), anti-DNA-PK (Cell Signaling, 12311), anti-TIPIN (Bethyl, A301-474A-1), anti-RPA1 (Santa Cruz, sc46504), anti-RPA2 (Millipore, 04-1481), anti-SSRP1 (Abcam, ab26212), anti-SPT16 (Cell Signaling, 12191), anti-SKP1 (Santa Cruz, sc-5281), and anti-phospho-H2AX (Millipore, 05-636).
6
Evaluation of Protein Regulation in RF-EMF Exposed Cells
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After exposure to RF-EMF, cells were harvested, and protein lysates were prepared as previously described [20 (link)]. Histones were extracted with 0.2 M HCl and neutralized with 1 M NaOH. Lysates or histones with 15–20 μg of protein were separated on 10–15% SDS polyacrylamide gels (PAGE) and electro-transferred to PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were incubated overnight at 4°C with primary antibodies including anti-phospho-Erk1/2, anti-Erk1/2, anti- β-actin, anti-histone3 (Cell signaling Technology, Inc.), and anti-phospho-H2AX (Millipore, Germany). After primary antibody incubation, membranes were then treated with the appropriate secondary antibodies for 1 h at room temperature. Protein bands were visualized by chemiluminescence detected with AmershamTM ImageQuant 800TM (Cytiva).
7
Apoptosis Signaling Pathway Analysis
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Cisplatin (HY-17394), Z-VAD-FMK (HY-16658), doxorubicin (HY-15142), and staurosporine (62996-74-1) were purchased from MCE (Middlesex, NJ, USA). ML216 was purchased from MCE (HY-12342) and Selleck (S0469, Houston, TX, USA). Hydroxyurea (V900323) and etoposide (E1383) were purchased from Sigma (St. Louis, MO, USA). The antibodies used included anti-DBC1 (Bethyl Labotatories, Montgomery, TX, USA, A300-434A), anti-V5 (Invitrogen, Waltham, MA, USA, R960-25), anti-Flag (Sigma, F7425), anti-BLM (Bethyl Labotatories, A300-110A), anti-GAPDH (Millipore, St. Louis, MO, USA, MAB374), anti-p21 (BD Biosciences, San Jose, CA, USA, 556430), anti-β Tubulin (Santa Cruz, Dallas, TX, USA, SC-166729), anti-p53 (Santa Cruz, SC-126), anti-Bcl-2 (Santa Cruz, SC-7382), anti-p16 (Santa Cruz, SC-56330), anti-phospho-H2A.X (Millipore, 07-164-25UG), anti-β-actin (Absin, Shanghai, China, ABS830031), anti-PARP1 (CST, Danvers, MA, USA, 9542S), anti-cleaved PARP1 (CST, 5625S), anti-BLM (Bioss, Beijing, China, bs-12872R) and anti-p21 (Bioss, bs-0741R). Secondary antibodies were horseradish peroxidase-coupled sheep anti-mouse IgG (GE Healthcare, Chicago, IL, USA, NA931 or ZSGB-BIO, Beijing, China, ZB-2301), donkey anti-rabbit IgG (GE Healthcare, NA934), and horseradish peroxidase-coupled sheep anti-mouse IgG (ZSGB-BIO, ZB-2305).
8
Antibody Panel for Cell Analysis
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The primary antibodies used in this study were as follows: anti-phospho-H2A.X and anti-Sox2 (Millipore, Billerrica, MA, USA), anti-p53 and anti-PARP (Cell Signaling Technology, Beverly, MA), anti-p21, anti-Slug and anti-Twist (Santa Cruz Biotechnology, Dallas, TX), anti-p16 and anti-Cyclin B1 (BD Biosciences, San Diego, CA), anti-Cyclin D1 (MBL International, Nagoya, Japan), anti-Nanog (ReproCELL, Yokohama, Japan), anti-b-actin and anti-Vinculin (SIGMA-Aldrich, St. Louis, MO).
9
Protein Extraction and Western Blot Analysis
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Proteins were extracted in RIPA buffer (Sigma, St. Louis, MO, USA) and quantified using Bradford assay (Bio-Rad, Hercules, CA, USA). Thirty micrograms of proteins were separated on 10-15% SDS-PAGE gel and transferred to Immobilion-P PVDF membranes (Millipore, Hayward, CA) and incubated with primary antibodies for anti-acetyl-histone H3 (06-599; 1:20000, Millipore), anti-phospho-H2A.X (07-164, 1:500, Millipore), anti-p53 (NCL-L-p53-DO; 1:1000, Novocastra, Newcastle, UK), anti-poli (ADP-ribose) polymerase (PARP, 11 835 238 001; 1:2000, Roche, Switzerland) and α-tubulin (CP06; 1:200, Millipore) (used as internal control).
Appropiate HRP-conjugated secondary antibody (P0260 or P0448; 1:2000; DAKO, Glostrup, Denmark) was used to reveal. Signals were developed with enhanced chemiluminiscence (Pierce, Rockford, IL).
Appropiate HRP-conjugated secondary antibody (P0260 or P0448; 1:2000; DAKO, Glostrup, Denmark) was used to reveal. Signals were developed with enhanced chemiluminiscence (Pierce, Rockford, IL).
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