Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 4 PARIS expression leads to p53 activation via PARIS Y137 phosphorylation-dependent epigenetic repression of MDM4. (A) Representative immunoblots examining the expression of MDM4, pS15-p53, p53 and FLAG (PARIS) in SH-SY5Y cells transfected with FLAG-PARIS wild-type or a Y137F mutant (48 h) using the indicated antibodies. b-Actin serves as an internal loading control. (B–D) Relative expression levels of MDM4 (B), p53 (C) and pS15-p53 (D) in the indicated experimental groups from A normalized to the internal loading control (b-actin; n = 3 per group). (E) Quantification of the relative expression of MDM4 mRNA in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h) deter- mined by RT-qPCR (normalized to internal GAPDH loading control; n = 3 per group). (F) Representative immunoblots of FLAG (PARIS) and MDM4 ex- pression in SH-SY5Y cells transfected (48 h) with mock or FLAG-PARIS and treated with TSA (300 nM, 42 h). (G) Quantification of the relative expression of MDM4 protein in the experimental groups in F normalized to b-actin (n = 3 per group). (H) A schematic diagram depicting the promoter structures of human MDM4 (hMDM4). IRS1, IRS2 and IRS3 motifs are indicated (top). Anti-acetyl-histone and anti-FLAG ChIP analysis of putative IRS (motif 1, 2 and 3) within the MDM4 promoter region in SH-SY5Y cells transfected with mock, or FLAG-PARIS wild-type (48 h, bottom) with or without the HDAC inhibitor TSA (300 nM, 42 h). The non-IRS region within the MDM4 promoter (Ctrl motif) was used as a negative control. Samples immuno- precipitated using either anti-histone antibodies or rabbit IgG were included as experimental controls in ChIP assays. (I) Quantification of relative ace- tylated histone enrichment on the indicated motifs located within MDM4 promoter determined by PCR amplification of ChIPed DNA in H (n = 3 per group). Data are expressed as mean SEM. *P 5 0.05, **P 5 0.01 and ***P 5 0.001 and statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Expressing, Activation Assay, Phospho-proteomics, Western Blot, Transfection, Mutagenesis, Control, Quantitative RT-PCR, Negative Control

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 5 Pharmacological inhibition of c-Abl activation restores behaviour/motor deficits and dopaminergic degeneration and prevents MDM4 repres- sion and p53 activation in mice with AAV-PARIS injection. (A) Representative exploratory paths from an open field test of mice that underwent AAV- Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). (B) Anxiety assessment of each experimental mouse group by examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 9 AAV-Con-injected mice, n = 10 AAV-Con-injected mice + nilotinib, and n = 8 AAV-PARIS-injected mice). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of vertical pole. (D) Motor coordination of each experimental mouse group used in B determined by the latency to fall in an accelerating rotarod test. (E) Representative TH immunohistochem- ical staining with Nissl counterstain of substantia nigra from mice that underwent AAV-Con or AAV-PARIS stereotaxic nigral injections (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The substantia nigra and ventral tegmental area regions are indicated by dot- ted yellow and white lines, respectively. Scale bar = 500 mm. (F) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) in the indicated mouse groups (n = 4 AAV-Con-injected mice + DMSO, n = 5 AAV-Con-injected mice + nilotinib and n = 4 AAV-PARIS-injected mice). (G) Representative TUNEL assay images of ventral midbrain from mice that underwent stereotaxic nigral injection of AAV-Con or AAV-PARIS (3 weeks) and treatment with the c-Abl inhibitor nilotinib (50 mg/kg/day, i.p. 2 weeks). The coronal brain sections were counterstained with DAPI. (H) Quantification of the percentage of TUNEL-labelled cells in AAV-Con- or AAV-PARIS-injected ventral midbrain regions from mice with or without nilotinib treatment (n = 16 sections from four mice per group). (I) Representative immunoblots examining pY137-PARIS, PARIS, c-Abl, pY245-c-Abl, MDM4, pS15-p53 and p53 expression in the ventral midbrain of AAV-Con- or AAV-PARIS-injected mice with or without nilotinib treatment using the indicated antibodies. (J) Quantification of the relative expression of pY137-PARIS, PARIS, c-Abl, pY245-c-Abl MDM4, pS15-p53 and p53 proteins nor- malized to b-actin (n = 5 AAV-Con-injected mice and n = 4 AAV-PARIS-injected mice). Data are expressed as mean SEM. Statistical analyses was per- formed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. **P 5 0.01 and ***P 5 0.001. DMSO = dimethyl sulphoxide; WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activation Assay, Injection, Staining, TUNEL Assay, Western Blot, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 6 Pharmacological inhibition of c-Abl activity in in vivo adult parkin knockout mice prevents motor dysfunction and dopaminergic neurode- generation with concomitant blocking of PARIS phosphorylation and p53 activation. (A) Representative exploratory paths from an open-field test of 6-month-old wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 m) and treated with the c-Abl in- hibitor nilotinib (200 mg nilotinib per 1 kg diet, p.o. for 2 months) or standard chow diet (chow). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border versus the sum of the centre and periphery zones (n = 4 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 4 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) that expe- rienced stereotaxic nigral injection of AAV-GFPCre with or without nilotinib treatment (200 mg nilotinib per 1 kg diet, p.o. for 2 months). The coronal brain sections were counterstained with DAPI. Magnified images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL- labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice with or without nilotinib treatment (n = 16 sections from four mice per group). (H) Representative immunofluorescence images examining the expression of pY245-c-Abl in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treat- ment. (I) Quantification of the relative pY245-c-Abl fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (J) Representative immunofluorescence images examining the expression of pY137-PARIS in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (K) Quantification of the relative pY137-PARIS fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). (L) Representative immunofluorescence images examining the expression of pS15-p53 in TH-stained dopamine neurons from the AAV-GFPCre-injected ventral midbrain regions of wild-type littermate and parkinfl/flmice with or without nilotinib treatment. (M) Quantification of the relative pS15-p53 fluorescence signal in the ventral midbrain regions of the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Inhibition, Activity Assay, In Vivo, Knock-Out, Blocking Assay, Phospho-proteomics, Activation Assay, Injection, Immunohistochemical staining, Staining, TUNEL Assay, Expressing, Two Tailed Test

Journal: Brain : a journal of neurology

Article Title: Parkin interacting substrate phosphorylation by c-Abl drives dopaminergic neurodegeneration.

doi: 10.1093/brain/awab356

Figure Lengend Snippet: Figure 7 Y137F-PARIS-mediated suppression c-Abl-PARIS pathway rescues MDM4 repression, blocks p53 activation and prevents development of motor deficits and dopamine neuron loss in parkin knockout mice. (A) Representative exploratory paths from an open field test of 6-month-old wild- type littermate or homozygous floxed parkin mice (parkinfl/fl) nigrally injected with AAV-GFPCre (3 months) AAV-PARIS-Y137F (3 months, phospho- deficient mutant PARIS). (B) Anxiety assessment of each experimental mouse group examining the percentage of exploration time in the border ver- sus the sum of the centre and periphery zones (n = 8 mice per group). (C) Pole test for motor function assessment of each experimental mouse group used in B examining the latency to reach the base of the vertical pole (n = 8 mice per group). (D) Representative TH immunohistochemical staining of substantia nigra from wild-type littermate or homozygous floxed parkin mice (parkinfl/fl) with intranigral injection of AAV-GFPCre AAV-PARIS-Y137F. Scale bar = 500 mm. (E) Stereological assessment of TH-positive dopaminergic neurons in the SNpc (injection side) of the indicated mouse groups (n = 4 mice per group). (F) Representative TUNEL assay images of ventral midbrain from wild-type littermate or homozygous floxed parkin mice (par- kinfl/fl) that experienced stereotaxic nigral injection of AAV-GFPCre AAV-PARIS-Y137F. The coronal brain sections were counterstained with DAPI. Merged images are shown in the bottom panel. (G) Quantification of the percentage of TUNEL-labelled cells in AAV-GFPCre-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice AAV-PARIS-Y137F (n = 16 sections from four mice per group). (H) Representative immunoblots examining the expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53, and parkin in the AAV-GFPCre AAV-PARIS-Y137F-injected ventral midbrain regions from wild-type littermate and parkinfl/flmice using the indicated antibodies. b-Actin serves as an internal loading control. (I) Quantification of the relative expression of pY245-c-Abl, c-Abl, pY137-PARIS, PARIS, MDM4, pS15-p53 and parkin proteins normalized to b-actin in the indicated experimental groups (n = 4 mice per group). Data are expressed as mean SEM. Statistical analysis was performed using an ANOVA test followed by Tukey’s post hoc analysis or an unpaired two-tailed Student’s t-test. ***P 5 0.001. WT = wild-type.

Article Snippet: The following primary antibodies were used: rabbit GFP antibody (Cell Signaling Technology; Cat No 2956; 1:5000), mouse GFP antibody (Santa Cruz Biotechnology, Cat No sc9996, 1:500), mouse FLAG antibody (Sigma; Cat No F1804; 1:50 for immunoprecipitation), rabbit PARIS (ZNF746) antibody (Proteintech; Cat No 24543-1-AP; 1:5000), rabbit antibody to phosphorylated c-Abl (Cell Signaling Technology; Cat No 2868; 1:5000), mouse c-Abl antibody (Sigma; Cat No A5844; 1:5000), rabbit MDM4 antibody (Proteintech; Cat No 17914-1-AP; 1:5000), mouse antibody to phosphorylated p53 (Cell Signaling Technology; Cat No 9284; 1:5000), mouse p53 antibody (Santa Cruz Biotechnology; Cat No sc126; 1:5000), mouse PGC-1a antibody (Calbiochem, Cat No ST1202), rabbit NRF1 antibody (Abcam, Cat No ab34682), rabbit tyrosine hydroxylase (TH) antibody (Novus Biologicals; Cat No NB300-109; 1:2000), mouse TH antibody (ImmunoStar; Cat No 22941; 1:2000), mouse parkin antibody (Cell Signaling Technology; Cat No 4211; 1:5000), rabbit acetyl-histone H3 antibody (Merck Millipore; Cat No 06-599; 1:20), rabbit Histone H3 antibody (Cell Signaling Technology; Cat No 4620; 1:20), horseradish peroxidase (HRP)-conjugated mouse FLAG antibody (Sigma; Cat No 8592; 1:5000), HRPconjugated mouse HA antibody (Cell Signaling Technology; Cat No 2999; 1:5000), and HRP-conjugated b-actin mouse antibody (Sigma; Cat No A3854; 1:10 000).

Techniques: Activation Assay, Knock-Out, Injection, Mutagenesis, Immunohistochemical staining, Staining, TUNEL Assay, Western Blot, Expressing, Control, Two Tailed Test

Journal: Mechanisms of ageing and development

Article Title: Cellular senescence in vascular wall mesenchymal stromal cells, a possible contribution to the development of aortic aneurysm.

doi: 10.1016/j.mad.2021.111515

Figure Lengend Snippet: Fig. 5. (A and B) representative images of immunofluorescence labeling of p – H2AX in h – MSCs and AAA – MSCs isolated from two different donors. A FITC conjugated secondary antibody was used to detect the nuclear localization of p – H2AX. AAA – MSCs nuclei showed a stronger green fluorescent signal compared to h – MSCs nuclei (magnification 600X; bar: 100 nm); (C) representative western blot images showing total p53 and P-p53 protein expression in h – MSCs and AAA – MSCs. Results from two different donors were shown. (D) Relative amounts of total p53 and P-p53 protein expression were normalized to the intensity of actin and represented as fold increase relative to h – MSCs of each donor. Western blotting was performed in duplicate and the relative quantification was expressed as mean value ± SD. * represents a significant difference compared to h - MSCs, p < 0.05 (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Article Snippet: The membranes were blocked with dry milk (blocking reagent) (Invitrogen, Thermo Fisher Scientific, Monza, Italy) for 30 min at room temperature and were then incubated with the following primary antibodies: mouse anti-human p53 antibody (Cell Signaling Technologies, Euroclone, Milan, Pero); mouse anti-human phospho - p53 (Ser 15) (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human p21CIP antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti human p16INK4A antibody (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human Beclin (Cell Signaling Technologies, Euroclone, Milan, Italy); rabbit anti-human LC3 (Cell Signaling Technologies, Euroclone, Milan, Italy); mouse anti-human tubulin antibody (Sigma- Aldrich, St Louis, Missouri, USA) and mouse anti-human actin antibody (Millipore Merck, Darmstadt, Germany).

Techniques: Immunofluorescence, Labeling, Isolation, Western Blot, Expressing, Quantitative Proteomics

Journal: PloS one

Article Title: Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.

doi: 10.1371/journal.pone.0088140

Figure Lengend Snippet: Figure 4. ATM knocked down reversed Cuc B induced G2/M phase arrest. A549 cells transfected with 100 pmol ATM siRNA or negative control siRNA for 12 h followed by treatment with or without 200 nM Cuc B for another 24 h. The cell cycle was analyzed (A, B). A549 cells transfected with ATM siRNA and treated with or without 200 nM Cuc B for 24 h. Phosphorylation of Chk1 on Ser-345, Cdc25C on Ser-216, p53 on Ser-15 and protein levels of p53 Cyclin B1 were analyzed by Western blot (C). Total Cyclin B1 protein was immunoprecipitated from ATM siRNA transfected and treated with or without 200 nM Cuc B A549 cells lysates and analyzed for Cdk1 for 24 h (D). *p,0.05 vs. Cont. Cont, control group. doi:10.1371/journal.pone.0088140.g004

Article Snippet: Specific antibodies PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88140 against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phosphop53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA).

Techniques: Transfection, Negative Control, Phospho-proteomics, Western Blot, Immunoprecipitation, Control

Journal: PloS one

Article Title: Cucurbitacin B induced ATM-mediated DNA damage causes G2/M cell cycle arrest in a ROS-dependent manner.

doi: 10.1371/journal.pone.0088140

Figure Lengend Snippet: Figure 7. Schematic representation of the signaling pathways involved in the Cuc B induced G2/M checkpoint in A549 cells. Cuc B induced ROS formation, which lead to DNA damage. In response to DNA damage, ATM via autophosphorylation activated G2/M checkpoint. The downstream effectors of ATM, Chk1 and p53, were both activated, which resulted in Cdc25C phosphorylation and increased expression of 14-3-3-s respectively. Phosphorylation of Cdc25C on Ser-216 by activated Chk1 facilitated the binding of 14-3- 3-s to Cdc25C, which prevents Cdk1 dephosphorylation, and keeps Cdk1-Cyclin B1 complex inactivated, causing G2/M phase arrest. doi:10.1371/journal.pone.0088140.g007

Article Snippet: Specific antibodies PLOS ONE | www.plosone.org 2 February 2014 | Volume 9 | Issue 2 | e88140 against GAPDH, phospho-Cdc25C-Ser-216, Cdc25C, phosphop53-Ser-15, phospho-p53-Ser-20, p53, phospho-STAT3-Tyr-705, STAT3, phospho-ATM-Ser-1981, phosphor-ATR-Ser-428, ATR, phospho-Chk1-Ser-345, Chk1, phosphor-Chk2-Thr-68, Chk2 were purchased from Cell Signaling Technology (USA).

Techniques: Protein-Protein interactions, Phospho-proteomics, Expressing, Binding Assay, De-Phosphorylation Assay

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a Recombinant human PEPD (10–2560 nM) was incubated with recombinant human p53 or p53 mutants (250 nM) overnight at 4 °C. Their binding was measured by ELISA. Each value is mean ± SD ( n = 3). b Recombinant human PEPD (801 nM) and p53 (114 nM) or p53 R175H (114 nM) were incubated alone or with each other for 15 min at room temperature, then incubated with BS3 (a cross-linker) for 30 min at room temperature, and analyzed by WB for PEPD, p53 and p53 R175H . p53 was included in each experiment for comparison.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Recombinant, Incubation, Binding Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a WB analysis of p53, p53 mutants and PEPD in WCL of untreated cells. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a loading control. b , c PEPD, p53 or p53 mutant in a sample (WCL, cytosol or nuclear extract prepared from untreated cells) was completely pulled down by IP. An isotype-matched IgG was used as a control. Percentages of p53 or p53 mutant unbound to PEPD or vice versa were determined by measuring them in the supernatants using ELISA. The precipitates and an input were analyzed by WB for p53, p53 mutant and PEPD (Supplementary Figs. and ), and the percentages of p53 or p53 mutant bound to PEPD or vice versa were determined by comparing their band intensities with that of the inputs using ImageJ. d Levels of p53, p53 mutant and PEPD in WCL of untreated cells, measured by ELISA. The bar-dot plots in b – d show individual values and mean ± SD ( n = 3). p53 was included in each experiment for comparison.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Control, Mutagenesis, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a Cells were treated by siRNA (10 nM) for 72 h. Cell viability were measured by trypan blue assay. b Cells were treated by siRNA (10 nM) for 48 h. p53 and other proteins in WCL were analyzed by WB. c Cells were treated by siRNA (10 nM) for 48 h. PEPD, PEPD-p53 complexes, and PEPD-p53 mutant complexes in WCL were pulled down by PEPD IP, and the supernatant was analyzed for p53, p53 mutants and PEPD by WB. WCL of untreated MDA-MB-231 cells was used as a PEPD control in WB. d , e Cells were transfected with or without PEPD G278D , and 24 h later treated by vehicle or PEPD siRNA (10 nM) for 72 h. Cell viability was measured by trypan blue assay in d . PEPD and other proteins in WCL were analyzed by WB in e . Cells carrying p53 (MCF-7 cells and CAL-51 cells) or MDA-MB-231 (p53 KO ) cells were included in a – c for comparison. GAPDH is a loading control in b , c , and e . The bar-dot plots in a and d show individual values and mean ± SD ( n = 3). *** P < 0.001, **** P < 0.0001, ns—not significant, by analysis of variance followed by Tukey test in a or by paired two-tailed t -test in d .

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Control, Transfection, Comparison, Two Tailed Test

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a MDA-MB-231 (p53 KO ) cells were transfected with or without a p53 mutant for 48 h. p53 mutant and other proteins in WCL were analyzed by WB. b , c MDA-MB-231 (p53 KO ) cells were transfected with a p53 mutant and 24 h later treated with siRNA (10 nM) for 96 h. The relatively long siRNA treatment time was for harvesting enough cells for analysis. Cell viability was measured by trypan blue assay in b . p53 mutant and other proteins in WCL were analyzed by WB in c . d MDA-MB-231 DKO cells (p53 KO and PEPD KO ) were cotransfected with a p53 mutant and Tet-on-PEPD and treated with or without Dox (50 ng/ml) for 48 h. p53 mutant and other proteins in WCL were analyzed by WB. e , f MDA-MB-231 DKO cells were transfected with a p53 mutant and Tet-on-PEPD, treated with Dox (50 ng/ml) for 24 h, washed, and cultured with or without Dox (50 ng/ml) for 72 h. p53 mutant and other proteins in WCL were analyzed by WB in e . Cell viability was measured by trypan blue assay in f . GAPDH is a loading control in a and c–e . The bar-dot plots in b and f show individual values and mean ± SD ( n = 3). **** P < 0.0001, by paired two-tailed t -test.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Transfection, Mutagenesis, Cell Culture, Control, Two Tailed Test

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a Cells were treated by siRNA (10 nM) for 48 h. p53, p53 mutants and other proteins in subcellular fractions were analyzed by WB, using voltage-dependent anion channel (VDAC), GAPDH and lamin B as loading controls. b Cells were treated by siRNA (10 nM) for 48 h. MMP was measured by JC-1 fluorescence. c Cells were treated by siRNA (10 nM) for 48 h, from which mitochondria were isolated and analyzed by IP-WB for CYPD binding to p53 mutants. d Cells were treated by siRNA (10 nM) for 72 h. Apoptosis was measured by TUNEL assay, counting 500 cells per sample (see also Supplementary Fig. ). The bar-dot plots in b and d show individual values and mean ± SD ( n = 3). **** P < 0.0001, by paired two-tailed t -test in b . Cells carrying p53 (MCF-7 cells and CAL-51 cells) or MDA-MB-231 (p53 KO ) cells were included in a, b , and d for comparison.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Fluorescence, Isolation, Binding Assay, TUNEL Assay, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a Cells were treated by siRNA (10 nM) for 24 h. Phospho-p53 and phospho-p53 mutants in WCL were analyzed by WB. GAPDH is a loading control. b Cells were treated by siRNA (10 nM) for 48 h. Phospho-p53 and phospho-p53 mutants in subcellular fractions were analyzed by phostag WB. c Cells were transfected with an equal amount of PG13-Luc or MG15-Luc along with pRL-TK and 24 h later treated with siRNA (10 nM) for 48 h. Luciferase activity was measured in WCL. d Cells were treated by siRNA (10 nM) for 48 h. Binding of p53 mutants to the p53-binding sites in the promoters of CDKN1A and BBC3 genes were measured by ChIP-qPCR assay. The bar-dot plots in c and d show individual values and mean ± SD ( n = 3). **** P < 0.0001, by paired two-tailed t -test. Cells carrying p53 (MCF-7 cells and CAL-51 cells) or MDA-MB-231 (p53 KO ) cells were included in a–c for comparison.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Control, Transfection, Luciferase, Activity Assay, Binding Assay, ChIP-qPCR, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a, b Cells were treated by siRNA (10 nM) for 48 h. Ac-K373-p53 mutants in WCL in a and in subcellular fractions in b were analyzed by WB. c Cells were treated by siRNA (10 nM) for 48 h. PEPD-p53 mutant complexes were removed from the nuclear extracts by PEPD IP. The precipitate and the supernatant were analyzed by WB for p53 mutants and Ac-K373-p53 mutants. d , e Cells were treated by siRNA (10 nM) with or without C646 (8 μM) for 72 h. Cell viability was measured by trypan blue assay in d . PEPD and other proteins in WCL were analyzed by WB in e . f–i MDA-MB-231 (p53 KO ) cells were transfected with p53 R175H/K373R or p53 R280K/K373R and 24 h later treated by siRNA (10 nM) for 96 h. The relatively long siRNA treatment time was for harvesting enough cells for analysis. Cell viability was measured by trypan blue assay in f and h . PEPD and other proteins in WCL were analyzed by WB in g and i . A p21-positive sample (WCL of SK-BR-3 cells treated by PEPD siRNA) was used as a control in g . GAPDH is a loading control in a , e , g , and i . α-Tubulin, lamin B and VDAC are loading controls and for ruling out cross contamination in b . The bar-dot plots in d , f , and h show individual values and mean ± SD ( n = 3). * P < 0.05, ** P < 0.01, **** P < 0.0001, by paired two-tailed t -test.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Mutagenesis, Transfection, Control, Two Tailed Test

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a Recombinant p53 and p53 R175H were subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate for p53 and p53 R175H . b – f WCL of untreated cells in b , c , and e or WCL of cells treated with siRNA (10 nM) for 48 h in d and f were subjected to IP by PAb1620, PAb240, or isotype-matched IgG, followed by WB analysis of the precipitate for p53 mutants and PEPD. A p53-containing sample (WCL of untreated CAL-51 cells) was used as a positive control during WB in b . The relative level of each p53 mutant in d–f was measured by ImageJ.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Recombinant, Positive Control, Mutagenesis

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a , b Cells were treated by siRNA (10 nM) for 48 h with or without C646 (8 μM). WCL was subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate for p53 mutants. c, d MDA-MD-231 (p53 KO ) cells were transfected with p53 R175H/K373R, p53 R248Q/K373R , p53 R273H/K373R , or p53 R280K/K373R and 24 h later treated with siRNA (10 nM) for 48 h. WCL was subjected to IP by PAb1620 or PAb240, followed by WB analysis of the precipitate by p53 mutants and PEPD. A p53-containing sample (WCL of untreated CAL-51 cells) was used as positive control during WB in c . e , f MDA-MD-231 (p53 KO ) cells were transfected with p53 R175H/K373R or p53 R280K/K373R and 24 h later treated with siRNA (10 nM) for 48 h. p53 mutants in subcellular fractions was analyzed by WB. Lamin B, α-tubulin and VDAC were measured as loading controls and for ruling out cross contamination.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Transfection, Positive Control

Journal: Communications Biology

Article Title: Loss of peptidase D binding restores the tumor suppressor functions of oncogenic p53 mutants

doi: 10.1038/s42003-021-02880-x

Figure Lengend Snippet: a–i Mice bearing orthotopic breast tumors generated from MDA-MB-231 (p53 R280K ) cells, MDA-MB-231 (p53 KO ) cells, HCC70 (p53 R248Q ) cells, or MDA-MB-231 (p53 R175H ) cells were treated by intratumor injection of siRNA (10 pmol) every 3 days. Arrows in a , c , e , and g indicate treatment start. The experiments were stopped 24 or 48 h after the last siRNA dose. Treatment of HCC70 (p53 R248Q ) tumors was suspended for 7 days before the final dose, because the tumors in the PEPD siRNA group became too small to treat. Each value in a – h is mean ± SEM ( n = 13–16). The bar plots in b , d , f , and h show individual values and mean ± SEM ( n = 13–16). **** P < 0.0001, by paired two-tailed t -test. PEPD and other proteins in tumor homogenates (two tumors per group) were analyzed by WB ( i ). GAPDH is a loading control. j Graphic representation of reactivation of p53 mutants by PEPD KD. A PEPD homodimer binds to a homo-tetramer of p53 mutants. p53 mutants freed from PEPD due to PEPD KD undergo K373 acetylation and other PTM (mono-ubiquitination and phosphorylation), which result in refolding of the mutants and reactivation of their transcription-dependent and transcription-independent tumor-suppressing activities.

Article Snippet: Anti-BAK (Cat# 3792), anti-BAX (Cat# 2772), anti-BCL-2 (Cat# 2870), anti-BCL-XL (Cat# 2764), anti-BID (Cat# 2002), anti-β-tubulin (cat#86298), anti-cleaved caspase 3 (Cat# 9661), anti-cleaved caspase 7 (Cat# 9491), anti-cleaved caspase 8 (Cat# 9496), anti-cleaved caspase 9 (Cat# 9501), anti-Cyto c (Cat# 4272), anti-EGFR (Cat# 2232), anti-EndoG (Cat# 4969), anti-MKK3 (Cat# 8535), anti-MYC (Cat# 9402), anti-p53 (Cat# 2524, 2527 and 18032), anti-phospho-p53 (S6) (Cat#9285), anti-phospho-p53 (S15) (Cat# 9284), anti-phospho-p53 (S20) (Cat# 9287), anti-phospho-p53 (S46) (Cat#2521), anti-p21 (Cat# 2946), and anti-VDAC (Cat# 4866) were purchased from Cell Signaling Technology.

Techniques: Generated, Injection, Two Tailed Test, Control, Ubiquitin Proteomics, Phospho-proteomics

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