Activation of p53 induces the expression of various gene products, which in turn can either prevent proliferation of damaged cells or induce apoptosis, thereby removing damaged cells from the body. 22 Phosphorylation of H2AX plays a major role in the DNA damage response and is required for the assembly of DNA repair proteins. 23 Therefore, we investigated these molecules, given their role in apoptosis and DNA damage. 9, 23 For analysis of the xenograft model, selected tumors excised from the treated and control mice were stained for anti-pH2AX and anti-p53 (Cell Signaling Technology). To further investigate the effect of EDO-S101 on DNA damage, rat spleen sections (10 mm) were also stained for anti-pH2AX and anti-p53 (Cell Signaling Technology). For analysis of the vasculitis model, kidney sections (4 mm) were stained with periodic acid-Schiff and hematoxylin and eosin stains to assess morphology as previously described. [19] [20] [21] 24 In addition to renal pathology, rats with EAV develop pulmonary vasculitis and hemorrhage as previously described 25 (see Supplementary Methods for further details).
Anti ph2ax
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-pH2AX is a laboratory reagent used to detect the phosphorylated form of the histone protein H2AX. Phosphorylation of H2AX is an early cellular response to DNA double-strand breaks, making Anti-pH2AX a valuable tool for the study of DNA damage and repair processes.
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Lab products found in correlation
Anti cleaved parp, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti p akt, by Cell Signaling Technology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti caspase 3, by Cell Signaling Technology (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 568 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
N sim super resolution microscope, by Nikon (1 mentions)
Nis elements software, by Nikon (1 mentions)
Anti lamin b1, by Abcam (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti beclin 1, by Santa Cruz Biotechnology (1 mentions)
Anti atg12, by Cell Signaling Technology (1 mentions)
Anti atg7, by Abcam (1 mentions)
Anti p62, by BD (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Anti lc3b, by Cell Signaling Technology (1 mentions)
Lsm510 meta confocal laser microscope, by Zeiss (1 mentions)
21 protocols using anti ph2ax
1
Investigating DNA Damage and Apoptosis
2
Mitochondrial Morphology Analysis in Hematopoietic Stem Cells
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MPPs or LT-HSCs were sorted into the slide coated with poly-l -lysine (SIGMA). Cells were placed on ice for 30 min to permit cells to settle onto the slide and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were then permeabilized with 0.2% TritonX-100 for 15 min at room temperature and blocked with 5% goat serum for 1 h at room temperature. Cells were incubated with the anti-p-H2A.X (Cell Signaling Technology, #9718, 1:200) or anti-Tom20 (Santa Cruz Biotechnology, sc-11415, 1:500) antibody overnight at 4 °C, washed and incubated in the Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen, A11011, 1:1000) for 1 h at room temperature. Cell nuclei were stained with DAPI and mounted using fluorescence mounting medium (DAKO). Images were acquired with a Confocal Microscope A1R or super resolution microscope N-SIM (Nikon), and were processed with NIS-Elements software (Nikon). For quantitative analysis of mitochondrial morphology, images were first thresholded and then converted to binary images by using ImageJ software69 (link),70 (link). Individual particle (mitochondria) were analyzed for mitochondrial area and number of fragments.
3
Autophagy Markers Quantification via Western Blot
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Cell lysis, SDS-page and Western blotting were performed according to standard procedures as described previously [45 (link)]. The following antibodies were used: anti-Beclin-1 (Santa Cruz Biotechnology, Heidelberg, Germany, No. sc-48381), anti-Atg12 (Cell Signaling Technology, Danvers, MA, USA, No. 2010), anti-Atg7 (Abcam, Cambridge, USA, No. ab53255), anti-LC3b (Cell Signaling Technology, No. 3868), anti-p62 (Becton Dickinson, Heidelberg, Germany, No. 610832), anti-Tubulin (Sigma, St. Louis, MO, USA, No. T8203-25UL), anti-Lamin B1 (Abcam, No. ab16048), anti-cleaved PARP (Cell Signaling Technology, No. 5625), anti-CyclinD1 (Cell Signaling Technology, No. 2926) and anti-pH2AX (Cell Signaling Technology, No. 9718).
In order to quantify protein expression, we used ImageJ® (by Wayne Rasband at NIH, Bethesda, MD, USA) for densitometric analysis as described previously [49 (link)]. In brief, band density was measured relative to the untreated control and then adjusted to tubulin as loading control. For analysis of isolated cytosolic and nuclear fractions, tubulin and Lamin B1 were utilized as loading controls and to demonstrate proper separation.
In order to quantify protein expression, we used ImageJ® (by Wayne Rasband at NIH, Bethesda, MD, USA) for densitometric analysis as described previously [49 (link)]. In brief, band density was measured relative to the untreated control and then adjusted to tubulin as loading control. For analysis of isolated cytosolic and nuclear fractions, tubulin and Lamin B1 were utilized as loading controls and to demonstrate proper separation.
4
Immunohistochemical Analysis of Alzheimer's Pathology
5
Protein Extraction and Western Blot
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Preparation of samples: total cell protein extracts were separated by 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto polyvinylidene difluoride membranes (PVDF, Thermo Fisher Scientific). Anti-ß-actin antibody (AC-15) was purchased from Sigma, anti-rad51 (#8875), anti-rad52 (#3425), anti-p21 (#2947) and anti-p-H2AX (#9718) from Cell Signaling Technology, and anti-p53 (DO-1) from Thermo Fisher Scientific and anti-MVP (#ALX-801–005) from Enzo Life Science. Secondary anti-mouse (#7076) and anti-rabbit (#7074) horseradish peroxidase-labeled antibodies were obtained from Cell Signaling Technologies.
6
Western Blot Analysis of Signaling Proteins
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Protein lysates (20–30 μg) were loaded onto 10–12% SDS-PAGE gels, electrophoresis, and blocking were conducted as described16 (link). Blots were probed overnight at 4 °C with respective antibodies. Primary antibodies (anti-p-Akt, anti-p-EGFR, anti-Bcl-XL, anti-Ku80, anti-p-STAT3, STAT3, anti-p-H2AX, and anti-β-actin) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-EGFR and anti-Rad51 antibody was purchased from Santa Cruz Biotechnology, Dallas, TX, USA, and anti-EphB4 antibody (clone m265) was provided by Vasgene Therapeutics Inc. (Los Angeles, CA, USA). Horseradish peroxidase (HRP)–conjugated secondary antibodies were obtained from Sigma (St. Louis, MO, USA).
7
Immunoblotting Analysis of Stem Cell Markers
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Immunoblotting were performed as previously described45 (link). The protein content was determined using Bradford Reagent (#5000006; Bio‐Rad) and the antibodies used were purchased from Cell Signaling: anti-caspase3 (#9662) and anti-pH2A.X (#9718); Millipore: anti-nestin (#MAB5326), anti-CD133 (#MAB4399), and anti-SOX2 (#AB5603); or from Santa Cruz Biotechnology (Dallas, USA): anti-GFAP (#sc-6171-R). ImageJ bundled with Java 1.8.0_172 (U. S. National Institutes of Health, Maryland, USA) was used to normalize intensity levels according to loading controls expression blotted with anti‐GAPDH (#AM4300; ThermoFisher Scientific) or anti‐β‐actin (#sc-69879; Santa Cruz Biotechnology). Original immunoblots are represented in Suppl. Figure S11.
8
Western Blot Analysis of Cochlear Proteins
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Whole cochlear pooled protein extracts were prepared from 3 mice, as described [35 (link)]. A fixed volume of extract was separated by SDS-PAGE and transferred to PVDF membranes (0.2 μm, Bio-Rad Laboratories, Hercules, CA, USA) using the Bio-Rad Trans Blot TURBO apparatus. Prior to antibody incubation (overnight 4 °C), membranes were blocked with 5% bovine serum albumin or non-fat dried milk in 0.1% Tween, 1 mM TBS. Primary antibodies used were as follows: rabbit anti-P-p38 (1:1000, #9211), anti-P-JNK (1:1000, #4668), anti-P-AKT (1:1000, #9271) and anti-P-H2AX (1:1000, #2577) (all from Cell Signaling Technology, Danvers, MA, USA), rabbit anti-P22phox (1:250, #sc-20781; Santa Cruz Biotechnology, Dallas, TX, USA), anti-HO-1 (1:1000, #AB1284; Merck, Darmstadt, Germany), anti-MnSOD (1:1000, #06-984; Millipore, Merck, Darmstadt, Germany), goat anti-NQO1 (1:1000, #ab2346; Abcam) and rabbit anti-PI3K (1:15000, in-house). Immunocomplexes were visualized with peroxidase-conjugated secondary antibodies (1 h at room temperature), and bands were detected using the Clarity™ Western ECL Substrate (Bio-Rad) on an Image Quant LAS4000 mini digital camera (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Band densities were quantified in triplicate using Image Quant TL software 8.1 (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA ).
9
Western Blot Analysis of Protein Markers in Transgenic Mice Lung Tissues
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Proteins from transgenic mice lung tissues or whole cell extractions were quantified using the BCA assay (Thermo Scientific, Rockford, IL). A total of 50 μg protein extracts were subjected to electrophoresis on a NuPAGE 10% Bis-Tris gel (Invitrogen) and transferred to a polyvinylidene difluoride (PVD) membrane by iBlot gel transfer system (Invitrogen, Grand island, NY). The membranes were blocked for 1 h with 5% non-fat dry milk in TBST (10 mM Tris–HCl, pH 7.4, 100 mM NaCl, 0.1% (V/V) Tween −-20), and successively incubated with primary antibodies overnight at 4 °C. Anti-EGFP (Sigma, St. Louis, MO) and anti-DNMT3B (Imgenex, San Diego, CA, USA) antibodies were used to detect DNMT3B expression in transgenic mice lung tissue, while β-actin was used as an internal control. DNA chromosome aneuploidy related proteins were probed using the following antibodies: anti-pH2Ax, anti-53BP and anti-pCHK2 purchased from Cell Signaling technology (Danvers MA), and Anti-Mad2L2 obtained from Abcam (Cambridge MA). The reaction was followed by probing with peroxidase-coupled secondary antibodies including anti-mouse IgG or anti-Rabbit IgG at dilution 1:5000 (Santa Cruz Biotechnology, Dallas, TX, USA), binding results were visualized using enhanced chemiluminescence (GE Healthcare).
10
Comprehensive Antibody Panel for Signaling Pathway Analysis
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Antibodies against AKT, ERK 1/2, phospho-ErbB3 (Y1289), phospho-AKT (S473), phospho-ERK 1/2 (T202/204), anti-pH2A.X, anti-pH3 and anti-cleaved-PARP were purchased from Cell Signaling Technology. Anti-ErbB3, anti-P27, anti-Cyclin D1, anti-CDK4, anti-UBI and anti-GAPDH were obtained from Santa Cruz Biotechnology. Anti-rabbit and anti-mouse were purchased from AbCam. Anti ErbB3 antibodies A3 and A4 have been described previously [28 (link)–30 (link)]. The two Anti-ErbB3 antibodies are all of the IgG1 isotype (EM unpublished observation). Vemurafenib and trametinib were obtained from Selleck Chemicals. TaqMan probes for ErbB3, NRG1 and housekeeping gene 18S were purchased from Applied Biosystems. The rabbit anti-Ki67 polyclonal antibodies were from Zymed Laboratories. FITC-conjugated goat anti-mouse IgG was obtained from Cappel Research Products. Texas Red-conjugated goat anti-rabbit IgG were from Jackson Immunoresearch Laboratories. DAPI was purchased from Sigma. Vybrant DiI cell labeling solution was from Invitrogen. LysoTracker-Red was obtained from Molecular Probes.
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