Journal: Scientific reports

Article Title: MLKL is involved in the regulation of skin wound healing and interplay between macrophages and myofibroblasts in mice.

doi: 10.1038/s41598-025-97729-2

Figure Lengend Snippet: Fig. 3. MLKL is involved in inflammatory mediator synthesis and apoptosis in wound site. (A) The skin wound tissues of MLKL+/+ and MLKL−/− mice were subjected to immunofluorescence staining for Caspase-3 (red), Bcl-2 (green) P53 (green) and nuclear (DAPI, blue) on the 5, 7 and 10th days after wound injury. Merge represents the composite picture of target protein and nuclear. The confocal microscope was used for image acquisition (×200 magnification), and the mean value of fluorescence intensity was used to perform calculation (n = 6). (B) The protein expression of Caspase-3, Bcl-2 and P53 in wound tissue of MLKL+/+ and MLKL−/− mice was evaluated by western blot (n = 4–6). GAPDH was used as a loading control. Grayscale values were measured using ImageJ software. (C) The secretion of IL-6, TNF-α and PGE2 in wound tissue and serum of MLKL+/+ and MLKL−/− mice was evaluated by ELISA (n = 6). Littermate control mice were on a C57BL/6J genetic background were utilized in the experiments. Results were expressed as the mean ± SD of multiple independent experiments and analyzed by Student t test. *P < 0.05, **P < 0.01, ***P < 0.001. a. u. arbitrary unit. Scale label = 20 μm.

Article Snippet: Name Description KDa Concentration Company Cat no. ERα Mouse monoclonal 71.4 kDa 1:250 GeneTex GTX13538 MMP-9 Rabbit polyclonal 73 kDa 1 ug/mL Novus NBP2-41233 VEGF Rabbit polyclonal 42 kDa 1 ug/mL Novus NB100-2381 arginase Mouse monoclonal 35 kDa 1:200 Santa SC-47,715 Ym1 Rabbit polyclonal 45 kDa 1:1000 Stemcell technologies 60,130 GAPDH Rabbit polyclonal 37 kDa 1:5000 Affinity AF7021 COX-2 Rabbit monoclonal 70 kDa 1:1000 Cell signaling technology D5H5 MLKL Rabbit polyclonal 54 kDa 1:1000 Affinity biosciences DF7412 Caspase-3 Rabbit polyclonal 17 kDa 1:500 abcam ab49822 Bcl-2 Mouse monoclonal 26 kDa 1:1000 Cell signaling technology 15,071 p53 Mouse monoclonal 53 kDa 1:1000 Cell signaling technology 2524T EGF Rabbit monoclonal 133 kDa 1:1000 abcam ab184266 Goat Anti-Rabbit IgG (H + L) HRP / 1:5000 Affinity biosciences S0001 Goat Anti-Mouse IgG (H + L) HRP / 1:5000 Affinity biosciences S0002 Table 3.

Techniques: Immunofluorescence, Staining, Microscopy, Fluorescence, Expressing, Western Blot, Control, Software, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: p53 Localization at Centrosomes during Mitosis and Postmitotic Checkpoint Are ATM-dependent and Require Serine 15 Phosphorylation

doi: 10.1091/mbc.E03-12-0900

Figure Lengend Snippet: Immunostaining of p53 phosphorylated at Ser15 and centrosomes (γ-tubulin) in AHH1 cells untreated (A), exposed to 20 mM NaF for 1 h (B), exposed to 0.2 μg/ml NOC for 20 h (C).

Article Snippet: Fixed and permeabilized cells were preincubated in 20% goat serum for 30 min at 37°C in a humidified chamber, and then incubated for 2 h with mouse monoclonal anti-p53 antibody (1:100 dilution, clone DO-7; DakoCytomation Denmark A/S, Glostrup, Denmark), rabbit anti-phospho-p53 (Ser15) antibody (1:2000 dilution, #9284; Cell Signaling Technology, Beverly, MA), rabbit anti-γ-tubulin antibody (1:2000 dilution, T3559; Sigma-Aldrich, St. Louis, MO), all in 5% goat serum.

Techniques: Immunostaining

Journal:

Article Title: p53 Localization at Centrosomes during Mitosis and Postmitotic Checkpoint Are ATM-dependent and Require Serine 15 Phosphorylation

doi: 10.1091/mbc.E03-12-0900

Figure Lengend Snippet: (A) Western blot analysis of phospho-ser15-p53 (for details, see MATERIALS AND METHODS) in untreated cultures or after exposure to 0.2 μg/ml NOC for 20 h. (B) Cell-cycle distribution as measured by FACS analysis of DNA content versus BrdU incorporation of asynchronously growing untreated cultures (CTR), after exposure to 0.2 μg/ml NOC for 20 h (NOC) or at different times of release (rel.) from NOC-induced arrest. (C) Western blot analysis of cyclin B expression in the same experimental conditions as in B.

Article Snippet: Fixed and permeabilized cells were preincubated in 20% goat serum for 30 min at 37°C in a humidified chamber, and then incubated for 2 h with mouse monoclonal anti-p53 antibody (1:100 dilution, clone DO-7; DakoCytomation Denmark A/S, Glostrup, Denmark), rabbit anti-phospho-p53 (Ser15) antibody (1:2000 dilution, #9284; Cell Signaling Technology, Beverly, MA), rabbit anti-γ-tubulin antibody (1:2000 dilution, T3559; Sigma-Aldrich, St. Louis, MO), all in 5% goat serum.

Techniques: Western Blot, BrdU Incorporation Assay, Expressing

Journal: Oncotarget

Article Title: Singular v Dual inhibition of SNF2L and its isoform, SNF2LT, have similar effects on DNA Damage but opposite effects on the DNA Damage Response, Cancer Cell Growth Arrest and Apoptosis

doi:

Figure Lengend Snippet: MDA-MB-468 cells were transfected with the different siRNAs. A, singular knockdowns of either SNF2L or SNF2LT both led to substantial increases in p53 mRNA but dual knockdowns affected p53 mRNA less so by real time RT-PCR. B, singular knockdowns of either SNF2L or SNF2LT both led to substantial increases in the p53 target gene, 14-3-3σ but dual knockdowns did not affect 14-3-3σ. C, singular knockdowns of either SNF2L or SNF2LT both led to substantial increases in another p53 target gene, GADD45A but dual knockdowns did not affect GADD45A. Each experiment was performed in triplicate and repeated at least four times.

Article Snippet: Antibodies used for detection of DNA damage were obtained from the DNA Damage Antibody Sampler (Cell Signaling Technology, Inc., Danvers, MA), which included Phospho-ATR (Ser428), Phospho-ATM (Ser1981) mouse monoclonal antibody (mAb), Phospho-BRCA1 (Ser1524), Phospho-CHK1 (Ser296), Phospho-CHK2 (Thr68), Phospho-Histone H2AX (Ser139), and Phospho-p53 (Ser15; 16G8) mouse mAb. p53 (7F5) rabbit mAb and β-actin (13E5) rabbit mAb were also used (Cell Signaling Technology, Inc.).

Techniques: Transfection, Quantitative RT-PCR

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Increased DSB and DDR are associated with cellular senescence in BRAP-deficient cells and cerebral cortices (A) Representative images of phospho-p53 immunofluorescence staining of WT and Brap −/− MEFs at P2. (B) Immunoblotting of total protein extracts from WT and Brap −/− MEFs at P1 and P3, respectively. (C) Immunoblotting of DNA damage response (DDR) proteins in WT and Brap −/− MEFs at P1. (D) Representative images of double immunofluorescence staining of WT and Brap −/− MEFs at P3 with antibodies against Ki67 (red) and 53BP1 (green). (E) Representative images of double immunofluorescence staining of WT and Brap −/− MEFs at P3 with antibodies against Ki67 (green) and γH2A.X (red). (F) Immunoblotting analyses of histone extracts of MEFs at P1 or P3, showing increased γH2A.X and γH2A.X mono-ubiquitination in Brap −/− MEFs. (G) Kaplan–Meier curve shows significantly shortened lifespan ( p < 0.0001 by log rank test) of Brap cKONPC mice ( n = 46) relative to their littermate control mice ( n = 29). (H) Representative images of senescence-associated β-gal analysis of cerebral cortical sections from Brap cKONPC and control mice at three months of age. Note the pyramidal neuronal morphology of some SA-β-gal + cells (red arrows) in high magnification views. (I) RT-qPCR shows an increased expression of p16 Ink4a and Pai-1 (plasminogen activator inhibitor-1/Serpine1) in three-month-old Brap cKONPC cortices (Mean ± SD; n = 4 biological replicates). p-values calculated by Student’s t test are indicated. (J) Immunoblotting of cerebral cortical total protein extracts, demonstrating higher levels of senescence and SASP-like changes in the cortical tissue of Brap cKONPC relative to littermate control mice. (K and L) Immunoblotting of histone extracts from cortical tissue of three-month-old Brap cKONPC and control mice, showing elevated γH2A.X as well as the presence of mono- and poly-γH2A.X ubiquitination in Brap cKONPC mice (boxed bands). K and L are the same immunoblot that was first probed by anti-γH2A.X, stripped, and re-probed by anti-ubiquitin. (M) Immunoblotting of total protein extracts from cortical tissue of Brap cKONPC and control mice, showing persistent elevation of 53BP1 in Brap cKONPC cortices from P8 (postnatal day 8) to 12M (12 months). (N) Representative images of γH2A.X-NeuN double immunohistological staining of cerebral cortical sections from 4-month old WT or Brap cKONPC mice. The presence of γH2A.X immunoreactivity in Brap cKONPC neurons is indicated by arrows. (O) Immunoblotting of total protein extracts from cortical tissue of Brap cKONPC or control mice at various ages, showing that Brap LOF does not increase cleaved caspase 3, an apoptosis marker. Embryonic Lis1 +/− and Nde1 −/− cortical tissues were used as positive controls for the presence of apoptosis. Nuclear DNA was stained with Hoechst 33,342. Bars: 50 um or as indicated.

Article Snippet: Phospho-p53 (Ser15) (D4S1H) , Cell Signaling Technology , Cat# 12571; RRID: AB_2714036.

Techniques: Immunofluorescence, Staining, Western Blot, Double Immunofluorescence Staining, Ubiquitin Proteomics, Control, Quantitative RT-PCR, Expressing, Marker

Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet:

Article Snippet: Phospho-p53 (Ser15) (D4S1H) , Cell Signaling Technology , Cat# 12571; RRID: AB_2714036.

Techniques: Ubiquitin Proteomics, Recombinant, Protease Inhibitor, Plasmid Preparation, Knock-Out, Generated, Software

Journal: Oncogene

Article Title: Proteomic identification of p53-dependent protein phosphorylation.

doi: 10.1038/onc.2008.124

Figure Lengend Snippet: Figure 1 Induction and phosphorylation of p53 and apoptosis in mitomycin C (MMC)-treated HCT116 cells. (a) HCT116 p53 þ / þ and p53/ cells were treated with MMC for 16 and 24 h. Whole-cell lysates were analysed for p53, p21, MDM2 and phosphorylation of p53 at Ser15, Ser20 and Ser392 by western blotting. b-Actin expression was used as loading control. (b) Immunostaining of HCT116 p53 þ / þ and p53/ cells after 16 h treatment with MMC. Phosphorylation of p53 was detected by antibodies specific for phosphorylated Ser15 and Ser37. (c). Apoptosis was assessed by fluorescence-activated cell sorting (FACS) analysis of caspase activity in HCT116 p53 þ / þ and p53/ cells treated with MMC for 16 and 24 h. HCT116 p53 þ / þ and p53/ colon carcinoma cells (provided by Bert Vogelstein, Johns Hopkins Oncology Center, Baltimore, MD, USA) were grown in Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% fetal bovine serum, glutamin and gentamycin in 5% CO2 at 37 1C. HCT116 cells were grown with or without 10 mg/ml mitomycin C to activate endogenous p53 (Sigma, St Louis, MI, USA). Western blotting was performed according to standard procedure using the following primary antibodies: mouse monoclonal anti-p53 (DO-1, Eurogenetics, Tessenderlo, Belgium), mouse monoclonal anti- MDM2 (2A10, provided by Sonia Lain, University of Dundee), mouse monoclonal anti-p21 (BD Pharmingen, San Diego, CA, USA), mouse monoclonal anti-b-actin (Sigma, St Louis, MI, USA). For detection of phosphorylation of p53, rabbit polyclonal anti-p53 Ser15, Ser20 and Ser392 (Cell Signaling Technology, Danvers, MA, USA) were used. Immunofluorescence staining was performed as described (Rahman et al., 2005). Caspase activity was measured using CaspaTag Pan caspase kit (Chemicon, Temecula, LA, USA).

Article Snippet: For detection of phosphorylation of p53, rabbit polyclonal anti-p53 Ser15, Ser20 and Ser392 (Cell Signaling Technology, Danvers, MA, USA) were used.

Techniques: Phospho-proteomics, Western Blot, Expressing, Control, Immunostaining, FACS, Activity Assay, Staining

Journal: Developmental biology

Article Title: Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

doi: 10.1016/j.ydbio.2019.01.011

Figure Lengend Snippet: Fluorescent Immunohistochemistry

Article Snippet: Mouse monoclonal anti-p53 (phospho Ser15) 16G8 , Cell Signaling , Cat#9286; RRID:AB_331741.

Techniques: Transduction, Recombinant, Imaging, Binding Assay, Software

Journal: Developmental biology

Article Title: Epithelial DNA methyltransferase-1 regulates cell survival, growth and maturation in developing prostatic buds

doi: 10.1016/j.ydbio.2019.01.011

Figure Lengend Snippet: (A) RT-PCR for Cdkn1a mRNA in E18.5 control and cDnmt1KO urethras. (B-C) E18.5 urethra sections were labeled with antibodies against Gamma-H2AX (in red, DNA damage marker) and LacZ (in green, labels Shh lineage epithelium). (D-E) E18.5 urethra sections were labeled with antibodies against Phospho-p53 Ser15 (in red, marks active p53). (F-G) E18.5 urethra sections were labeled with antibodies against Cleaved caspase 3 (in red, marks apoptotic cells) and EYFP (in green, labels Shh lineage epithelium). Green channel is excluded for ease of visualization. (H) Percentage of cleaved Caspase 3 labeled cells in the Shh lineage urethral epithelium. White arrowheads indicated cleaved Caspase 3 positive apoptotic cells. White dotted lines indicate epithelial-mesenchymal interface. DAPI staining is shown in blue. Scale bar is 50 microns. Graphical results are the mean + SEM of at least three mice per group. p-values indicate significant differences (* p<0.05, ** p<0.01) between groups based on Student’s unpaired t-test.

Article Snippet: Mouse monoclonal anti-p53 (phospho Ser15) 16G8 , Cell Signaling , Cat#9286; RRID:AB_331741.

Techniques: Reverse Transcription Polymerase Chain Reaction, Control, Labeling, Marker, Staining