Journal: Nucleic Acids Research

Article Title: Site-specific acetylation of polynucleotide kinase 3′-phosphatase regulates its distinct role in DNA repair pathways

doi: 10.1093/nar/gkae002

Figure Lengend Snippet: List of antibodies used in this study

Article Snippet: phospho-53BP1 , Cell Signaling Technology , 2675 , 1:500.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: The Functional Characterization of GCaMP3.0 Variants Specifically Targeted to Subcellular Domains

doi: 10.3390/ijms23126593

Figure Lengend Snippet: Sub-cellular distribution of transiently expressed GCaMP3.0 variants . Human embryonic kidney cells (HEK293) were either non-transfected with a GCaMP3.0 encoding construct ( A1 – E1 ) or with GCaMP3.0cyto ( A2 – E2 ), GCaMP3.0pm ( A3 – E3 ), GCaMP3.0mito ( A4 – E4 ), GCaMP3.0mom ( A5 – E5 ), or GCaMP3.0nuc ( A6 – E6 ). Expression of GCaMP3.0 variants was detected by their green fluorescence ( A2 – A6 ). For identificationof the Golgi apparatus in cells, they were transfected with Golgi-RFP ( B1 – B6 ). The endoplasmic reticulum was stained with primary rabbit anti-calnexin antibodies (dilution 1:100) and secondary donkey anti-rabbit-A594 (dilution 1:1000) antibodies ( C1 – C6 ). Labeling of mitochondria was performed with MitoTracker DeepRed FM ( D1 – D6 ). Fluorescent images were obtained by confocal imaging of the samples. Composite images are depicted in ( E1 – E6 ). The scale bar denotes 8 µm.

Article Snippet: The following primary antibodies were used: rabbit (rb)-anti-calnexin (dilution 1:100; #2674, Cell Signaling Technology, Danvers, MA 01923, USA) and (rb)-anti-MAP2 (dilution 1:500; #AB5622, Chemicon/Millipore, Darmstadt, Germany).

Techniques: Transfection, Construct, Expressing, Fluorescence, Staining, Labeling, Imaging

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Influence of HO-1 induction on p38 MAPK activation in BeWo cells under T. gondii infection. (A,B) BeWo (1 × 10 6 cells/2,000 μl/well) cells were cultured in six-well plate during 24 h, infected with T. gondii (1:1) or not (control) for 3 h, treated with hemin (80 μM) or not (NaOH at 0.08% as vehicle) during 24 h, and submitted to western blotting assays for detection of phosphorylated p38 MAPK and β-actin. Protein bands were photodocumented, and densitometric analysis was performed by the ratio among phosphorylated p38 MAPK ( p -p38 MAPK) and its corresponding β-actin (endogenous control). (A) Representative blots for p -p38 MAPK and β-actin in the non-infected (NI) or infected ( T. gondii ) and hemin-treated or not (vehicle) BeWo cells. (B) Densitometric analysis for p -p38 MAPK. (C) BeWo (3 × 10 4 cells/200 μl/well) cells were cultured in 96-well plate during 24 h, pretreated with 10 μM of SB203580 (p38 MAPK inhibitor) or not (control) for 3 h, infected with T. gondii (1:1) for 3 h, and treated with hemin (80 μM) or not (control) during 24 h, and the T. gondii intracellular proliferation was analyzed by the β-galactosidase assay. The data were shown as number of tachyzoites (× 10 3 ). For both assays, the results were expressed as mean ± SEM from two or three independent experiments performed in six or eight replicates. Significant differences were statistically assessed by the Mann-Whitney test (B) or by one-way ANOVA with Bonferroni’s posttest (C) ; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Activation Assay, Infection, Cell Culture, Control, Western Blot, MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: Heme Oxygenase-1 Induction in Human BeWo Trophoblast Cells Decreases Toxoplasma gondii Proliferation in Association With the Upregulation of p38 MAPK Phosphorylation and IL-6 Production

doi: 10.3389/fmicb.2021.659028

Figure Lengend Snippet: Proposed mechanisms triggered by the HO-1 to control T. gondii infection by BeWo trophoblast cells. (A) Upper panel, a non-infected BeWo cell expresses normal levels of HO-1 and phosphorylated p38 MAPK, in addition to secreting baseline levels of IL-6. (B) Left panel, when BeWo cells were infected with T. gondii , the amounts of HO-1 and phosphorylated p38 MAPK were significantly decreased and the IL-6 levels were upregulated. (C) Right panel, when finally T. gondii -infected BeWo cells were treated with hemin, a specific HO-1 inducer, the parasite proliferation was significantly reduced, sequentially associated with an overexpression of HO-1, an increasing of the p38 MAPK phosphorylation and a releasing of high IL-6 levels, which was higher than those naturally secreted by the T. gondii -infected cells non-induced for HO-1 (left panel).

Article Snippet: Then, the membranes were blocked for 1 h with blotting buffer (25 mM Tris-HCl, 150 mM NaCl, 0.1% ( v / v ) Tween 20, and pH 7.4) added with 4% non-fat dry milk (Molico, Nestlé ® , São Paulo, SP, Brazil) and incubated, overnight, with the following primary antibodies diluted in a blotting buffer with 2% non-fat dry milk: goat polyclonal anti-human-HO-1 (1:1,000; Santa Cruz Biotechnology Cat# sc-1797, Santa Cruz, CA, United States), rabbit polyclonal anti-human-phospho-p38 MAPK (T 180 /Y 182 ) (1:400; R&D Systems Cat# AF869, Minneapolis, MN, United States), or mouse monoclonal anti-human-β-actin (1:1,000; Santa Cruz Biotechnology Cat# sc-81178).

Techniques: Control, Infection, Over Expression, Phospho-proteomics

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: PP4R3β S840 phosphorylation is required for 53BP1 recruitment to damaged chromatin. a MS/MS spectrum of the PP4R3β phospho-peptide harboring S840. Phosphorylated residue is highlighted in green. b Co-immunoprecipitation of 53BP1 or PP4C with PP4R3β S840 phospho-variants. HeLa cells transiently transfected with the indicated FLAG-53BP1 and Myc-PP4R3β constructs were left in an asynchronous state (Asyn) or synchronized to prometaphase (M) by treatment with nocodazole (Supplementary Fig. ). Whole cell extracts were immunoprecipitated (IP) with indicated agarose beads and analyzed by immunoblot using indicated antibodies. EV: empty vector; S/D: S840D; S/F: S840F; S/A: S840A. c Immunoblots showing the kinetics of 53BP1 phosphorylation throughout mitosis in cells expressing PP4R3β S840 phospho-variants . HeLa cells were transfected with the indicated siRNAs and complemented with the indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. Transfected cells were released from prometaphase arrest and harvested at indicated time points for immunoblot analysis using indicated antibodies. Phospho-histone H3 at Ser10 (H3pS10) indicates mitotic state. d Irradiation-induced 53BP1 foci formation in cells expressing PP4R3β S840 phospho-variants. Mitotic HeLa cells were complemented with Myc-PP4R3β S840 phospho-variant constructs as described in c , collected from prometaphase arrest and seeded on poly-D-lysine-coated coverslips. Four hours after release, cells were irradiated with 5 Gy. Two hours post-irradiation, cells were fixed and analyzed by immunofluorescence with indicated antibodies. γ-H2AX staining marks sites of DNA damage. e Quantifications of 53BP1 foci in d , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. f Radiosensitivity of cells expressing PP4R3β S840 phospho-variants. HeLa cells were complemented with siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs as described in c . Viability was evaluated by clonogenic survival. Immunoblots confirm siRNA efficiency and expression of siRNA-resistant constructs. Data are expressed as mean ± s.d; n = 3. g Response of BRCA1-null UWB1.289 cells expressing PP4R3β S840 phospho-variants to indicated concentration of clinical-grade PARP inhibitor Olaparib. Cells were complemented as described in c . Viability was assessed by CellTiter-Glo colorimetric assay. Immunoblots confirm siRNA efficiency and expression of siRNA-resistant constructs. Data are expressed as mean ± s.d; n = 3

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Phospho-proteomics, Tandem Mass Spectroscopy, Residue, Immunoprecipitation, Transfection, Construct, Western Blot, Plasmid Preparation, Expressing, Variant Assay, Irradiation, Immunofluorescence, Staining, MANN-WHITNEY, Concentration Assay, Colorimetric Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: CDK5 is active in mitosis. a Phosphorylation of 53BP1 T1609/S1618 at distinct stages of mitosis and in G1. RPE1 cells were fixed at 0, 15, 30, 60, 90, and 120 min after release from G2/M arrest and stained with phospho-specific antibody against T1609/S1618 (pT1609/pS1618). Cells at indicated stages of mitosis were selected based on chromatin morphology, as indicated by DAPI staining. b Fluorescence intensity of phosphorylated 53BP1 over area of interest was quantified. Staining intensities from number of events per stage (indicated in the parenthesis) were expressed as mean ± s.d. A.U: arbitrary unit. c Phosphorylation of PP4R3β S840 at distinct stages of mitosis and in G1. RPE1 cells were synchronized and fixed at time points after release as described in a and stained with a phospho-specific antibody against S840 (pS840). d Fluorescence intensity of phosphorylated PP4R3β over area of interest was quantified as described in b . e Distribution of HeLa cells across distinct stages of mitosis at indicated time points after release from prometaphase arrest. HeLa cells were arrested at G2/M by RO-3306 and released into nocodazole to enrich for cells in prometaphase for 1 h. Prometaphase-arrested cells were collected by shake-off and released to progress through mitosis into G1 and fixed at indicated time points. Cells corresponding to stages of mitosis were tallied based on chromatin morphology, as indicated by DAPI staining. f Kinase activity levels of CDK5 in mitosis and G1. HeLa cells were synchronized and released as described in e . CDK5, immunoprecipitated from cells collected at indicated time points after release, was incubated with substrate peptide and [ 32 P]-ATP. Radioactivity of labeled substrate peptide was measured in a liquid scintillation counter. Data are expressed as mean ± s.d; n = 3. Representative immunoblots show indicated proteins present in the cell lysate for kinase assay. g Immunoblots showing the kinetics of 53BP1 T1609/S1618 phosphorylation and PP4R3β S840 phosphorylation throughout mitosis. HeLa cells were collected at indicated time points after release from synchronization as described in e and were analyzed with indicated antibodies

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Phospho-proteomics, Staining, Fluorescence, Activity Assay, Immunoprecipitation, Incubation, Radioactivity, Labeling, Western Blot, Kinase Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: PP4R3β is a bona fide substrate of CDK5. a Reconstituted CDK5/p25 complex was incubated with purified wild-type or S840A PP4R3β. 32 P-ATP signal indicates CDK5/p25-mediated phosphorylation. Ponceau-stained blots show input proteins. b HeLa cells stably expressing FLAG-HA empty vector (EV) or FLAG-HA PP4R3β were arrested in prometaphase, lysed, and immunoprecipitated for FLAG-HA PP4R3β. Elution from FLAG immunoprecipitation was analyzed by immunoblot with indicated antibodies. c RPE1 cells released from RO-3306-induced G2/M arrest were immediately treated with DMSO or 10 µM of CDK5 inhibitor compound 20-223. One hour after release from RO-3306, prometaphase cells were collected by shake-off and seeded on poly-D-lysine-coated coverslips. Three hours after release, cells were irradiated with 5 Gy IR. Two hours post-irradiation, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX staining marks sites of DNA damage. d Quantifications of 53BP1 foci in c , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. e Effect of CDK5 inhibition on radiosensitivity. Hela cells were treated with DMSO or 250 nM of 20-223 for 48 h, followed by irradiation at the indicated doses. Cell viability was evaluated by clonogenic survival. Data are expressed as mean ± s.d; n = 3. Immunoblots indicate abrogation of PP4R3β S840 phosphorylation from treatment with 20-223. f Response of BRCA1-null UWB1.289 cells treated with DMSO or 250 nM of 20-223 to indicated concentration of clinical-grade PARP inhibitor Olaparib. Cell viability was assessed by CellTiter-Glo assay. Data are expressed as mean ± s.d; n = 3. Immunoblots indicate abrogation of PP4R3β S840 phosphorylation from treatment with 20-223. g Melanoma A375 cells stably expressing CDK5 analog-sensitive variant (A375-AS) were arrested in prometaphase. Mitotic cells were released into media containing either DMSO or 10 µM of non-hydrolyzable ATP analog, 1NMPP1, to specifically inhibit CDK5. Cells were harvested at indicated time points for immunoblot analysis with indicated antibodies. h Quantification of immunoblot intensity in g , from duplicate repeat experiments, by ImageJ

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Incubation, Purification, Phospho-proteomics, Staining, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Irradiation, MANN-WHITNEY, Inhibition, Concentration Assay, Glo Assay, Variant Assay

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: CDK5 regulates dephosphorylation and the recruitment of 53BP1 to DNA damage. a Endogenous 53BP1 was immunoprecipitated from DMSO-treated or 1NMPP1-treated A375-AS cells and collected at indicated time points after release from arrest at prometaphase. Levels of co-immunoprecipitated endogenous PP4R3β were analyzed by immunoblot. b Endogenous 53BP1 was immunoprecipitated from DMSO-treated or 1NMPP1-treated A375-AS cells, collected at indicated time points, after release from nocodazole-induced arrest at prometaphase. Levels of 53BP1 T1609/s1618 phosphorylation from immunoprecipitated endogenous 53BP1 were analyzed by immunoblot. c Mitotic A375-AS cells, collected by shake-off from arrest at prometaphase, were immediately treated with DMSO or 10 µM of 1NMPP1 and seeded on poly-D-lysine-coated coverslips. 90 min after release, treated cells were irradiated with 5 Gy. 240 min after release, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX was stained to mark sites of DNA damage. d Quantifications of 53BP1 foci in c , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. e A375-AS cells were exposed to 0.2 µM Aphidicolin and DMSO or 10 µM 1NMPP1 for 12 h. Cells were fixed and stained for 53BP1 and Cyclin-A, a negative marker for G1 cells. f Quantification of percentage of cells containing 53BP1-containing nuclear bodies in e out of total cells, indicated in parenthesis, pooled from triplicate repeat experiments. P -values, Mann–Whitney U Test

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: De-Phosphorylation Assay, Immunoprecipitation, Western Blot, Phospho-proteomics, Irradiation, Staining, MANN-WHITNEY, Marker

Journal: Nature Communications

Article Title: A mitotic CDK5-PP4 phospho-signaling cascade primes 53BP1 for DNA repair in G1

doi: 10.1038/s41467-019-12084-x

Figure Lengend Snippet: A CDK5-PP4 signaling axis drives 53BP1 recruitment to damaged chromatin. a A375-AS cells were depleted of endogenous PP4R3β by siRNA knockdown and transfected with indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. Transfected cells were synchronized to prometaphase. Mitotic cells collected by shake-off were immediately treated with DMSO or 10 µM of 1NMPP1 and seeded on poly-D-lysine–coated coverslips. Ninety minutes after release, treated cells were irradiated with 5 Gy. 240 min after release, cells were fixed and stained with indicated antibodies to detect irradiation-induced 53BP1 foci. γ-H2AX was stained to mark sites of DNA damage. b Quantifications of 53BP1 foci in a , pooled from triplicate repeat experiments, expressed as mean ± s.d. Total number of cells is indicated in parenthesis. P -values, Mann–Whitney U Test. c A375-AS cells were depleted of endogenous PP4R3β by siRNA knockdown and transfected with indicated siRNA-resistant Myc-PP4R3β S840 phospho-variant constructs. The transfected cells were exposed to 0.2 µM Aphidicolin and DMSO or 10 µM 1NMPP1 for 12 h. Cells were fixed and stained for 53BP1 and Cyclin-A, a negative marker for G1 cells. d Quantification of percent cells containing 53BP1-containing nuclear bodies in c out of total cells (indicated in parenthesis) pooled from triplicate repeat experiments. P -values, Mann–Whitney U Test. e Model depicting CDK5 as a regulator of 53BP1 recruitment to damaged chromatin

Article Snippet: Rabbit polyclonal anti-pT1609/S1618-53BP1 antibody was produced by Antagene (Sunnyvale, CA) with a peptide, Cys-NRLREQYGLGPYEAV(p)TPLTKAADI(p)SLDN.

Techniques: Knockdown, Transfection, Variant Assay, Construct, Irradiation, Staining, MANN-WHITNEY, Marker