Journal: Molecular and Cellular Biology

Article Title: ΔNp73 Modulates Nerve Growth Factor-Mediated Neuronal Differentiation through Repression of TrkA

doi: 10.1128/mcb.02112-06

Figure Lengend Snippet: FIG. 4. Np73 is the predominant isoform in undifferentiated PC12 cells and is down-regulated upon NGF treatment. (A and B) Character- ization of p73 antibodies that can recognize exogenous murine Np73 (A) or Np73 (B). PC12 cells were uninduced or induced to express Np73 (A) or Np73 (B) for 18 h. Cell lysates were immunoprecipitated (IP) with 1 g of BL906 and p73Ab-2 antibodies, respectively, followed by Western blot (WB) analysis with p73-Ab1 (A) or p73-Ab3 (B). Approximately 2% of cell lysates was used as input. (C, D, and E) Characterization of endogenous p73 in PC12 and HCT116 cells. Cell lysates were immunoprecipitated with 1 g of BL906 and p73-Ab2 antibodies, respectively, followed by Western blot analysis with p73-Ab1 (C), p73-Ab3 (D), or BL906 (E). The HA-tagged murine Np73 (C, lane 2), Np73 (C and D, lane 1), and p73 (E, lane 2) as well as human TAp73 (E, lane 1) were used as controls. The asterisk in panel E, lane 3, represents a nonspecific protein. (F) Endogenous Np73 is down-regulated upon NGF treatment in PC12 cells. PC12 cells were treated with NGF or not treated with NGF for 0 to 72 h. Cell lysates were collected at each time point and analyzed by Western blot analysis with p73-Ab1, p73-Ab3, GAP-43, and actin antibodies, respectively.

Article Snippet: To do this, PC12 cells were uninduced or induced to express Np73 or Np73 for 18 h. Cell lysates were immunoprecipitated with BL906 antibody (Bethyl Laboratories, catalog no. A300-126A), which was raised against amino acids 1 to 62 of human TAp73 and expected to react with only the TAp73 isoforms, or p73-Ab2 antibody (Calbiochem, catalog no. 0P109), which was raised against amino acids 380 to 495 of human p73 and expected to detect both the and isoforms of TA/ Np73.

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: ΔNp73 Modulates Nerve Growth Factor-Mediated Neuronal Differentiation through Repression of TrkA

doi: 10.1128/mcb.02112-06

Figure Lengend Snippet: FIG. 7. Np73 directly represses the TrkA promoter. (A) Wild-type, but not mutant, TrkA promoter is suppressed by Np73. A schematic diagram of wild-type and mutant TrkA promoters is presented in the bottom panel. The dual-luciferase assay was performed as described in Materials and Methods. The increase in relative luciferase activity for each construct was calculated using an empty vector as a control. (B) Schematic presentation of the TrkA and p21 promoters with the locations of the transcriptional start site, p53-RE, and primers used for ChIP assays. (C and D) Np73 binds to the TrkA and p21 promoters in vivo. Cells were uninduced or induced to express Np73 (C) or Np73 (D) for 18 h followed by the ChIP assay as described in Materials and Methods. Anti-HA antibody (-HA) was used to immunoprecipitate Np73- or Np73-DNA complexes. Normal IgG was used as a control. (E) Endogenous Np73 binds to the TrkA promoter under a nonstressed condition in PC12 cells. p73-Ab1 and -3 antibodies were used to immunoprecipitate Np73-DNA complexes. Rabbit IgG and BL906 antibodies were used as a control.

Article Snippet: To do this, PC12 cells were uninduced or induced to express Np73 or Np73 for 18 h. Cell lysates were immunoprecipitated with BL906 antibody (Bethyl Laboratories, catalog no. A300-126A), which was raised against amino acids 1 to 62 of human TAp73 and expected to react with only the TAp73 isoforms, or p73-Ab2 antibody (Calbiochem, catalog no. 0P109), which was raised against amino acids 380 to 495 of human p73 and expected to detect both the and isoforms of TA/ Np73.

Techniques: Mutagenesis, Luciferase, Activity Assay, Construct, Plasmid Preparation, Control, In Vivo

Journal: Scientific reports

Article Title: Assessment of the toxic effect of benzalkonium chloride on human limbal stem cells.

doi: 10.1038/s41598-025-96919-2

Figure Lengend Snippet: Fig. 3. (A) Agarose gel electropherogram of amplification products (passage 1). Lane 1: DNA ladder (M), lanes 2–3: KRT3 (101 bp), lanes 4–5: KRT12 (190 bp), lane 6: DNA ladder (M), lanes 7–8: TP63 (96 bp), lanes 9–10: ABCG2 (144 bp), lanes 11–12: ACTβ (131 bp), lane 13: DNA ladder (M). NC, negative control. (B) Visualization of lack of expression of CK3, and CK12 and expression of positive markers p63 and ABCG2, in LSCs cultured in a medium supplemented with H/I/E. FITC-conjugated specific antibodies (green); DAPI- stained cell nuclei (blue). IC, isotype control. Scale bars = 15 μm.

Article Snippet: After rinsing with PBS, the cells were incubated with blocking buffer for 1 h, rinsed again, and labelled with rabbit anti-ABCG2 polyclonal (1:50; 27286-AP; Proteintech, USA), rabbit anti-p63 polyclonal (1:50; 12143-1-AP; Proteintech, USA), mouse anti-CK3 monoclonal (1:50; ab68260; Abcam, UK), and rabbit anti-CK12 monoclonal (1:50; ab185627; Abcam, UK) primary antibodies at 4 °C overnight.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, Expressing, Cell Culture, Staining, Control

Journal: Journal of Biological Chemistry

Article Title: Mst2 and Lats Kinases Regulate Apoptotic Function of Yes Kinase-associated Protein (YAP)

doi: 10.1074/jbc.m804380200

Figure Lengend Snippet: FIGURE5.YAP2isabletostabilizep73.A,bothWWdomainsofYAP2arerequiredtobindtop73.HA-p73with the indicated YAPs was transfected into HEK293 cells. Lysates were immunoprecipitated (IP) with FLAG anti- bodies,resolvedonSDS-PAGE,andimmunoblotted(IB)withHAantibodies(upperpanel).Theexpressionlevels of total HA-p73 (middle panel) and FLAG-tagged proteins (lower panel) were also monitored. B, time course of degradationofHA-p73inthepresenceofYAP2.HEK293cellsthatexpressYAP2WTinaninduciblesystemwere transfected with HA-p73. 24 h later, the cells were plated in fresh DMEM containing 1% FBS, blasticidin (5 g/ml), and zeocin (200 g/ml). Tetracycline (1 g/ml) was added to the medium to induce the expression of YAP2 WT. At 72 h and 96 h after induction, the cells were harvested followed by immunoblotting using antibodies of HA (upper panel), YAP (middle panel), or GAPDH (lower panel). C, the WW domains of YAP2 are required to stabilize the expression of p73. HEK293 cells that express control vector, YAP2 WT, YAP2 1&2 WW*, YAP2 S127A, or YAP1 WT in an inducible system were transfected with HA-p73 followed by analysis as described in B. Tetracycline was added to the medium for 96 h. D, YAP2 stabilizes p73. HA-p73 with the indicatedYAPswastransfectedintoHEK293cells,andeachplatewasdividedintotwo.24hlater,oneplatewas harvested, and 300 g/ml of cycloheximide (CHX; Sigma) was added to the other plate and harvested after additional 24 h. Cell lysates were immunoblotted using antibodies to HA (upper panel), YAP (middle panel), or GAPDH (lower panel). E, endogenous p73 was removed by p73-specific RNAi. Control RNAi oligo or p73-specific RNAi oligo was transfected to HEK293 cells that express YAP2 WT in an inducible system, and the expression of endogenous p73 was monitored by p73 antibody. F, removal of endogenous p73 weakens proapoptotic function ofYAP2.48hafterthetransfectionofRNAioligosinE,cellswerescatteredintofreshplatesandmaintainedinDMEM containing1%FCS.ExpressionofYAP2WTwasinducedbyaddingtetracycline.96hlater,thenumbersofcellswere counted, and expression of YAP2 WT was monitored as described in the legend for Fig. 3.

Article Snippet: Antibodies—HAantibody (Y-11), Lats1 antibody (N-18), p73 antibody (C-17), and cyclin E antibody (H-145) were purchased from Santa Cruz Biotechnology.

Techniques: Transfection, Immunoprecipitation, Expressing, Western Blot, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: Mst2 and Lats Kinases Regulate Apoptotic Function of Yes Kinase-associated Protein (YAP)

doi: 10.1074/jbc.m804380200

Figure Lengend Snippet: FIGURE 7. Schematic representation of the Hpo signaling pathway, which functions to promote survival of cells. All components investi- gated here are shown in solid gray with names in white letters and numbers. Other proteins depicted by symbols without any background and with black letters and/or numbers represent components shown previously as regulators of the Hpo pathway. K denotes kinase activity, and P in a circle denotes a phosphorylation site. Protruding triangles denote PPXY/PPXF motifs, and “cut out” triangles represent WW domains that recognize PPXY/PPXF motifs. WW45 acts as a scaffold protein for assembling Mst2 and Lats1 kinases. With the apparent help of WW45, Mst2 phosphorylates Lats1, which in turn phosphorylates YAP2 at Ser-127 (the same site on YAP1 is phosphorylated by Akt kinase (44)). Phosphorylated YAP2 is anchored in the cytoplasm by 14-3-3 protein. Apart from phosphorylation, Lats1 by itself can accumulate YAP2 in the cytoplasm. YAP2 free of phos- phorylation and association with Lats1 is able to enter the nucleus and promote transcription of proapoptotic genes. The YAP2-p73 complex sta- bilizes p73 and prevents its degradation, which is mediated by Itch, a competing WW domain-containing E3 ubiquitin ligase.

Article Snippet: Antibodies—HAantibody (Y-11), Lats1 antibody (N-18), p73 antibody (C-17), and cyclin E antibody (H-145) were purchased from Santa Cruz Biotechnology.

Techniques: Activity Assay, Phospho-proteomics, Ubiquitin Proteomics

Journal: Blood Cancer Journal

Article Title: Role of apurinic/apyrimidinic nucleases in the regulation of homologous recombination in myeloma: mechanisms and translational significance

doi: 10.1038/s41408-018-0129-9

Figure Lengend Snippet: a (I) Antibody array : Cell Cycle Antibody Array (Hypomatrix) was sequentially treated with ARP cell lysate and HRP-conjugated anti-APEX1 antibody. Interacting partners were then identified by their location on the array. (II) Immunoprecipitation: Interaction between endogenous APEX1 and p73 was detected in the ARP cells, either control (DMSO) or those treated with 5 µM API3 for 24 h. Nuclear lysates were immunoprecipitated using anti-P73 antibody or IgG (control) and protein complexes resolved on Western blot which was probed with anti-APEX1 antibody; Input, lysate before immunoprecipitation. b (I-III): APEX1 and P73 bind to RAD51 promoter in MM cells. Protein-DNA complexes from ARP cells treated as below were immunoprecipitated using anti-P73 antibody (I and III) or anti-APEX1 antibody (II). DNA was then extracted and occupancy of RAD51 promoter evaluated by Q-PCR; Treatments : C, control (DMSO); API3, cells treated with APEX1 inhibitor 5 µM for 24 h; CS, control shRNA; APEX1-KD, APEX1-knockdown cells; (IV) Western blot showing APEX1 in CS and APEX1-KD cells. c . Inhibition of APEX1 inhibits RAD51 promoter activity in MM cells. (I) RAD51 promoter was cloned upstream of firefly luciferase gene in a plasmid, which was then introduced into ARP cells. APEX1 in these cells was inhibited by treating them with small molecule (API3, 5 µM) for 24 h, and cells evaluated for luciferase activity; (II) ARP cells transduced with lentivirus particles carrying control (CS) or APEX1-shRNA (APEX1-KD) were selected in puromycin and following confirmation of knockdown, cells were co-transfected with RAD51 promoter plasmid (described in panel I) and a plasmid carrying Gaussia luciferase as control for transfection efficiency. RAD51 promoter activity was assessed from ratio of two luciferase activities; error bars represent SDs of triplicate assays

Article Snippet: For immunoprecipitation studies, anti-APEX1 antibody (Cat. # ab194, Abcam) and anti-p73 antibody (Cat. # NBP2-24737, Novus Biologicals) were used.

Techniques: Ab Array, Immunoprecipitation, Control, Western Blot, shRNA, Knockdown, Inhibition, Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Transduction, Transfection

Journal: iScience

Article Title: Targeting of Itch by clomipramine or gene therapy improves cognitive defects related to Alzheimer’s disease

doi: 10.1016/j.isci.2026.115181

Figure Lengend Snippet: Clomipramine inhibits Itch and prevents neuronal apoptosis and cell cycle re-entry in neurons from TgAD mice (A) Chemical structure of clomipramine. The chloride moiety is proposed to interact with catalytic cysteine in the HECT domain of Itch. (B) Rat cortical neurons were treated with Aβ 42 and/or clomipramine (75 nM) for 48 h followed by 12 h treatment with the proteasome inhibitor MG132. TAp73 was immunoprecipitated, and western blotting was performed on TAp73-IP with anti-ubiquitin and anti-Itch antibodies. Total protein lysates were also used for western blotting with indicated antibodies. Ubiquitinated TAp73 levels were quantified by densitometry of ubiquitin immunoblot, normalized with respect to the input TAp73, which was also normalized with respect to actin (mean ± SEM, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA, Tukey’s test, N = 3). (C) Rat cortical neurons were treated with Aβ 42 and/or 75 nM clomipramine for 48 h followed by western blotting for indicated proteins. The levels of TAp73, PCNA, and cl_caspase3 were quantified by densitometry, and fold change with respect to untreated (ctrl) neurons was determined (mean ± SEM, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, one-way ANOVA, Tukey’s test, N = 3). (D) Cortical neurons from WT or TgAD mice were treated with 75 nM clomipramine for 48 h followed by western blotting for indicated proteins. TAp73, PCNA, and cl_caspase3 levels were quantified by densitometry, and fold change with respect to untreated WT neurons is provided (mean ± SEM, ∗ p < 0.05, ∗∗ p < 0.01, one-way ANOVA, Tukey’s test, N = 2).

Article Snippet: PCNA (Santa cruz, cat. no. sc-56; 1:500 for WB, 1:50 for IHC), cleaved caspase 3 (CST, cat. no. 966l; 1:1000 for WB), Itch (CST, cat. no. 12117; 1:1000 for WB), TAp73 (Novus, cat. no. NBP2-24737, 1:1000 for WB, 1:50 for IP), myc-tag (9B11) (CST, cat. no. 2276; 1:2000 for WB, 1:100 for IHC), NeuN (Novus, cat. no. NBP2-67314; 1:100 for IHC), Ubiquitin (Santa cruz, cat. no. sc-8017; 1:1000 for WB) and β-Actin (Santa cruz, cat. no. sc-47778, 1:2000 for WB).

Techniques: Immunoprecipitation, Western Blot, Ubiquitin Proteomics

Journal: BMC Cancer

Article Title: Quercetin abrogates chemoresistance in melanoma cells by modulating ΔNp73

doi: 10.1186/1471-2407-10-282

Figure Lengend Snippet: Qct causes changes in p73 distribution . A) Non-quantitative PCR in DB-1 cells for TAp73 (Lane 1), ΔNp73 (Lane 2) and TAp73 positive control using SK Mel 28 cells (Lane 3) following treatment with TMZ. TAp73 was undetectable in the DB-1 cell line. B) Real time RT-PCR for ΔNp73 in DB-1 cell lines treated with TMZ 400 μM for 48 hrs followed Qct 75 μM for 24 hrs. Results are Mean ± SEM of triplicate experiments in DB-1 cell lines. C) p73 expression by western blot analysis in DB-1 and SK Mel 28 cell lines treated with vehicle (DMSO), TMZ 400 μM for 48 hrs followed by Qct 75 μM for 24 hrs. D) Immunocytochemical analysis of p73 localization in DB-1 cell lines treated with vehicle (DMSO), TMZ 400 μM for 48 hrs followed by Qct 75 μM for 24 hrs. Solid arrowheads indicate nuclear staining in cells without Qct treatment, while line-type arrowheads indicate cytoplasmic staining following Qct treatment.

Article Snippet: Antibody for GAPDH was purchased from Millipore-Chemicon (San Francisco, CA, USA). p73 antibody for western blotting and immunocytochemistry (ICC) was obtained from IMGENEX (San Diego, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: BMC Cancer

Article Title: Quercetin abrogates chemoresistance in melanoma cells by modulating ΔNp73

doi: 10.1186/1471-2407-10-282

Figure Lengend Snippet: Knockdown of p73 with siRNA restores PARP cleavage . Western blot analysis after transfection with p73 siRNA in DB-1 cell lines A) at basal level and B) after TMZ treatment and C) Quantification of p73 to GAPDH and Cleaved PARP relative to uncleaved PARP. D) Transient transfection of tyrosinase DNA in DB-1 cell lines and western blot analysis of p53 and 73 expression.

Article Snippet: Antibody for GAPDH was purchased from Millipore-Chemicon (San Francisco, CA, USA). p73 antibody for western blotting and immunocytochemistry (ICC) was obtained from IMGENEX (San Diego, CA, USA).

Techniques: Knockdown, Western Blot, Transfection, Expressing

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 1. p73 and - are phosphorylated during mitosis. A, mo- bility shift of p73 proteins in nocodazole-arrested cells. Hct116(3) cells were treated with hydroxyurea (2 mM) for 15 h and collected 1 and 6 h after wash-out to obtain cells enriched in S and G2 phases, respectively (43). The percentage of S phase cells was evaluated by bromodeoxyuri- dine incorporation (data not shown). Mitotic cells were obtained after 16 h of nocodazole treatment (50 ng/ml); G1-enriched cultures were collected 6 h nocodazole after release. nt, not treated (asynchronously growing untreated cells). Extracts were analyzed by immunoblotting using antibodies directed against the proteins, as indicated on the right (also see “Experimental Procedures”). B, mitotic mobility shift of p73 is due to phosphorylation. Extracts from either nocodazole-arrested (Noc, lanes 2 and 4) or interphasic (lanes 1 and 3) Hct116(3) cells were incubated at 30 °C for 1 h in the presence (lanes 3 and 4) or absence of -phosphatase (PPase) (lanes 1 and 2). Cell extracts were then analyzed by immunoblotting using 1288 anti-p73 antibody. C, p73 is hyperphos- phorylated in nocodazole- and taxol-arrested cells and in untreated mitotic cells. Hct116(3) cells were treated with either nocodazole (50 ng/ml, left panel) or taxol (1 M, middle panel) for 16 h. Nocodazole- arrested mitotic cells were separated from interphasic cells by vigorous shaking. Extracts prepared from asynchronously growing cells (lanes 1, 5, 7, and 8), total (lane 2), mitotic (lane 3), and interphase (lane 4) nocodazole-treated cells, taxol-treated cells (lane 6), and mitotic cells collected by shake-off from untreated asynchronously growing HCT116(3) cultures (lane 8) were analyzed by immunoblotting using anti-p73 (rows a and b) and anti-actin (row c) antibodies.

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques: Western Blot, Mobility Shift, Phospho-proteomics, Incubation

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 2. p73 protein associates with and is phosphorylated by p34cdc2-cyclin B complex. A, p73 is phosphorylated by the p34cdc2- cyclin B complex in vitro. Left panel, GST-p73 and GST were used as substrates for an in vitro kinase assay with recombinant p34cdc2-cyclin B complex. Middle panel, Ponceau Red staining of the same filter, showing the input GST proteins. Right panel, endogenous p73 and IKK proteins immunoprecipitated from HCT116(3) cells with the anti- p73 and anti-IKK antibodies, respectively, were subjected to an in vitro kinase assay with 20 units of purified recombinant p34cdc2-cyclin B complex (New England Biolabs) (upper right). The amount of recom- binant p34cdc2was evaluated by immunoblotting using anti-p34cdc2 an- tibody (lower right). B, the amino-terminal region of p73 (residues 1–120) is targeted by p34cdc2-cyclin B phosphorylation. GST and GST- p73 deletion mutants (maps in panel C) were subjected to in vitro kinase assay with purified recombinant p34cdc2-cyclin B complex. Be- low, the same filter was stained with Ponceau Red to visualize the input GST proteins used. C, schematic representation of GST-p73 mutant structure, phosphorylation and physical interaction with p34cdc2-cy- clin B. D, mapping of the phosphorylation target in p73. Left panel, in vitro kinase assay of GST-p73-(1–120) and GST-p73-(1–120)-T86A with 10 units of purified p34cdc2-cyclin B complex (upper panel). Input GST proteins were visualized by immunoblot (lower panel). Right panel, electrophoretic mobility of T86A p73 protein exogenously expressed in Hct116(3) cells is not affected by nocodazole treatment (50 ng/ml).

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques: In Vitro, Kinase Assay, Recombinant, Staining, Immunoprecipitation, Purification, Western Blot, Phospho-proteomics, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 3. p73 protein associates with the p34cdc2-cyclin B complex in mitosis. A, p73 binds to p34cdc2 in vivo, and this interaction increases in mitotic cells. Asynchronously growing or nocodazole-arrested Hct116(3) cells were detergent-solubilized and immunoprecipitated with either anti-p34cdc2 (lanes 2 and 4) or anti-HA tag (lanes 1 and 3) antibodies. Co-immunoprecipitated proteins (IP) were analyzed by immunoblotting with antibodies to p73, cyclin B, and p34cdc2. B, the physical association between p73 and p34cdc2-cyclin B decreases during exit from mitosis. Nocodazole-arrested Hct116(3) cells were allowed to resume mitosis after nocodazole removal. Cell extracts from asynchronously growing (A) or nocodazole-arrested (N) or -released cells after increasing times (Noc release) were immunoprecipitated with anti-p34cdc2 antibody, and the precipitates were analyzed by blotting using anti-p73, anti-cyclin B, and p34cdc2 antibodies. C, identification of the p34cdc2-interacting region of p73. 0.5 g of purified recombinant p34cdc2 (Santa Cruz) was incubated with GST alone, GST-p73, GST-p73, and GST-p73 deletion mutants followed by immunoblot (IB) analysis with anti-p34cdc2 antibody (upper panel). Bottom panel, the same filter was stained with Ponceau Red to visualize the input GST proteins in pull-down assays.

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Purification, Recombinant, Incubation, Staining

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 4. p34cdc2-cyclin B interferes with p73 functions. A, p34cdc2

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 5. p73 is displaced from mitotic chromosomes A and B. Subcellular localization of p73 in interphase and mitotic cells. Asyn- chronously cycling MCF7 cells (A) and ponasterone A (2.5 M)-induced H1299-derived p73 cells (B) were grown on glass coverslips for 24 h and then fixed and stained with anti-p73 antibody and rhodamine- conjugated secondary antibody. In row e, ponasterone A-induced H1299-derived p73 cells were exposed to nocodazole (50 ng/ml) for 16 h. Cells were counterstained with DAPI to detect DNA. p73 is pseudocolored in red (middle panel), whereas DNA appears in blue (left panel). C, p53 colocalizes with centrosomes in normal mitosis from asynchronously cycling ponasterone A-induced H1299-derived p53-in- ducible cells (row a). Cells were processed for immunofluorescence to -tubulin (rhodamine-conjugated secondary antibody, pseudocolored in red) and p53 (fluorescein isothiocyanate-conjugated secondary anti- body, pseudocolored in green) and counterstained with DAPI. After nocodazole (Noc) exposure for 16 h, the colocalization of p53 with -tubulin is lost (row b). ana, anaphase.

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques: Derivative Assay, Staining, Immunofluorescence

Journal: Journal of Biological Chemistry

Article Title: p73 Is Regulated by Phosphorylation at the G2/M Transition

doi: 10.1074/jbc.m304921200

Figure Lengend Snippet: FIG. 6. Differential behavior of p73 and p53 in response to spindle damage. A, p73-induced p21 expression decreases after no- codazole treatment. H1299-derived p73 inducible cells were either treated with ponasterone A (Pon A) (2.5 M) for 6 h (lanes 2 and 4) or mock-treated (lanes 1 and 3); nocodazole (Noc) (50 ng/ml) was then added to the culture medium for 18 h (lanes 3 and 4). Cell extracts were analyzed by immunoblotting using antibodies against the indicated proteins. The p21 signal was quantified by microdensitometry of auto- radiographs of ECL-processed immunoblots and normalized relative to the p21 signal measured in asynchronously cycling cells (lane 1). B, p21 levels increase after ponasterone A induction of p53 in nocodazole- treated H1299-derived p53-inducible cells. Cell extracts were prepared and analyzed as described in A.

Article Snippet: Cytological Preparations and Microscopy—Cells were fixed in 3.7% paraformaldehyde and processed for indirect immunofluorescence using antibodies against p73 (1:100, Imgenex) and p53 (1:200, Dako) -tubulin (3.5 g/ml in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) followed by Texas Red- and fluorescein isothiocyanateconjugated secondary anti-mouse IgG antibody (800-fold dilution in phosphate-buffered saline, 0.1% Tween 20, 5% fetal calf serum) before counterstaining in 0.1 g/ml DAPI.

Techniques: Expressing, Derivative Assay, Western Blot

Journal: FEBS open bio

Article Title: Methylation of drug resistance-related genes in chemotherapy-sensitive Epstein-Barr virus-associated gastric cancer.

doi: 10.1002/2211-5463.12765

Figure Lengend Snippet: Fig. 8. Gene expression in a metastatic lymph node lesion, measured using IHC in combination with hematoxylin. (A–E) High-magnification view of metastatic lymph node lesions and (F–J) positive control (ABCG2, AHNAK2, BCL2, FZD1, and TP73 IHC, 9200, scale bar 100 lm) are shown.

Article Snippet: ABCG2, AHNAK2, BCL2, FZD1, and TP73 staining of the metastatic lymph node sample was performed using anti-ABCG2 antibody (Clone B-1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-AHNAK2 antibody (Clone AG11779; Proteintech, Rosemont, IL, USA), anti-BCL2 antibody (Clone E17; Abcam), antiFZD1 antibody (Clone E-7; Santa Cruz Biotechnology), and anti-TP73 antibody (Clone EP436Y; Abcam) with detection carried out using DAB and Chromogen (DAKO, Glostrup, Denmark).

Techniques: Gene Expression, Positive Control

Journal: FEBS open bio

Article Title: Methylation of drug resistance-related genes in chemotherapy-sensitive Epstein-Barr virus-associated gastric cancer.

doi: 10.1002/2211-5463.12765

Figure Lengend Snippet: Fig. 9. Heatmap analysis of chemoresistance-associated gene methylation of EBVGC cases. The methylation patterns of each gene associated with chemoresistance, (A) ABCG2; (B) BCL2; (C) FZD1; (D) AHNAK2 and (E) TP73 were analyzed with heatmap and clustering analysis. The row indicates each case (EBV case; our case, rows which begin with TCGA; TCGA EBVGC cases), and the column indicates each probe corresponding to CpG sites in promotor region of each gene.

Article Snippet: ABCG2, AHNAK2, BCL2, FZD1, and TP73 staining of the metastatic lymph node sample was performed using anti-ABCG2 antibody (Clone B-1; Santa Cruz Biotechnology, Dallas, TX, USA), anti-AHNAK2 antibody (Clone AG11779; Proteintech, Rosemont, IL, USA), anti-BCL2 antibody (Clone E17; Abcam), antiFZD1 antibody (Clone E-7; Santa Cruz Biotechnology), and anti-TP73 antibody (Clone EP436Y; Abcam) with detection carried out using DAB and Chromogen (DAKO, Glostrup, Denmark).

Techniques: Methylation