rabbit polyclonal anti p p73  (Cell Signaling Technology Inc)

Journal: Molecular Medicine Reports

Article Title: The anticancer effects of cinobufagin on hepatocellular carcinoma Huh-7 cells are associated with activation of the p73 signaling pathway

doi: 10.3892/mmr.2019.10108

Figure Lengend Snippet: Cinobufagin may induce anticancer effects on Huh-7 cells via activation of p73 signaling. (A) Protein expression levels of p73, p-p73 (Y99), MDM2, p-MDM2 (S166), p21, Puma and Noxa in cells in cells following treatment with 5 µmol/l cinobufagin for 24 h, as determined by western blotting. Densitometric analysis of (B) p-p73, (C) Puma, (D) p-MDM2, (E) Noxa and (F) p21. (G) Expression of p73 in Huh-7 cells, as determined by immunocytochemistry. Representative images are shown at ×200 magnification. Data are presented as the means ± standard error of the mean of three independent experiments. *P<0.05 vs. control, # P<0.05 vs. cinobufagin. AURKA, aurora kinase A; MDM2, mouse double minute 2 homolog; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; p, phosphorylated; Puma, p53 upregulated modulator of apoptosis.

Article Snippet: The rabbit monoclonal anti-AURKA (cat. no. 14475), rabbit polyclonal anti-p-p73 (Y99; cat. no. 4665) and anti-p-MDM2 (S166; cat. no. 3521) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Activation Assay, Expressing, Western Blot, Immunocytochemistry, Control

Journal: Molecular Medicine Reports

Article Title: The anticancer effects of cinobufagin on hepatocellular carcinoma Huh-7 cells are associated with activation of the p73 signaling pathway

doi: 10.3892/mmr.2019.10108

Figure Lengend Snippet: Schematic diagram of cinobufagin-induced effects on hepatocellular carcinoma Huh-7 cells with mutant p53. Cinobufagin inhibits the viability, arrests the cell cycle and induces the apoptosis of Huh-7 cells by inhibiting AURKA and p53 signaling, and activating p73 signaling. AURKA, aurora kinase A; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma 2; CDK1, cyclin-dependent kinase 1; hnRNPK, heterogeneous nuclear ribonucleoprotein K; NKA, Na + /K + -ATPase; Noxa, phorbol-12-myristate-13-acetate-induced protein 1; p, phosphorylated; PCNA, proliferating cell nuclear antigen; Puma, p53 upregulated modulator of apoptosis.

Article Snippet: The rabbit monoclonal anti-AURKA (cat. no. 14475), rabbit polyclonal anti-p-p73 (Y99; cat. no. 4665) and anti-p-MDM2 (S166; cat. no. 3521) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA).

Techniques: Mutagenesis

Journal: The Febs Journal

Article Title: Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity

doi: 10.1111/febs.13108

Figure Lengend Snippet: Knockdown of p73 attenuates ADR-mediated induction of p53/TAp73-target gene expression. U2OS cells were transfected with control siRNA or with siRNA against p73 . Twenty-four hours after transfection, cells were treated with 0.5 μ m of ADR or left untreated. Twenty-four hours after treatment, whole cell lysates and total RNA were extracted and processed for (A) immunoblotting and (B) RT-PCR, respectively.

Article Snippet: Twenty-four hours after treatment, cells were washed twice in ice-cold PBS, fixed in 3.7% folmaldehyde in PBS at room temperature for 30 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and then blocked with 3% BSA in PBS at room temperature for 1 h. After blocking, cells were washed in ice-cold PBS and simultaneously incubated with mouse monoclonal anti-RUNX2 (8G5; Medical & Biological Laboratories, Nagoya, Japan) and rabbit polyclonal anti-p73 (Bethyl Laboratories, Montgomery, TX, USA) antibodies at room temperature for 1 h followed by the incubation with rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. After the extensive washing, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK).

Techniques: Knockdown, Targeted Gene Expression, Transfection, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: The Febs Journal

Article Title: Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity

doi: 10.1111/febs.13108

Figure Lengend Snippet: Complex formation between TAp73 and RUNX2 in response to ADR. (A) Nuclear co-localization of TAp73 with RUNX2 after ADR exposure. U2OS cells were exposed to 0.5 μ m of ADR (lower) or left untreated (upper). Twenty-four hours after treatment, cells were simultaneously incubated with monoclonal anti-RUNX2 (red) and polyclonal anti-p73 (green) antibodies. Cell nuclei were stained with DAPI (blue). Merged images (yellow) indicated the nuclear co-localization of TAp73 with RUNX2. Scale bar = 25 μm (magnification, × 630) (left). Percentages of RUNX2-, p73- or RUNX2/p73-positive cells were indicated (right). (B) Co-immunoprecipitation. An equal amount of whole cell lysates prepared from ADR-treated U2OS cells was immunoprecipitated with normal mouse serum (NMS) or with monoclonal anti-p73 antibody. The immunoprecipitates were analyzed by immunoblotting with the indicated antibodies (middle). The reciprocal experiments (right) and 1 : 20 inputs (left) are also shown.

Article Snippet: Twenty-four hours after treatment, cells were washed twice in ice-cold PBS, fixed in 3.7% folmaldehyde in PBS at room temperature for 30 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and then blocked with 3% BSA in PBS at room temperature for 1 h. After blocking, cells were washed in ice-cold PBS and simultaneously incubated with mouse monoclonal anti-RUNX2 (8G5; Medical & Biological Laboratories, Nagoya, Japan) and rabbit polyclonal anti-p73 (Bethyl Laboratories, Montgomery, TX, USA) antibodies at room temperature for 1 h followed by the incubation with rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. After the extensive washing, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK).

Techniques: Incubation, Staining, Immunoprecipitation, Western Blot

Journal: The Febs Journal

Article Title: Runt-related transcription factor 2 attenuates the transcriptional activity as well as DNA damage-mediated induction of pro-apoptotic TAp73 to regulate chemosensitivity

doi: 10.1111/febs.13108

Figure Lengend Snippet: RUNX2 represses the expression of p53/p73-target genes. (A) U2OS cells were transfected with or without the increasing amounts of the expression plasmid for RUNX2 (0.5, 1.0 or 2.0 μg). Forty-eight hours after transfection, whole cell lysates and total RNA were isolated and analyzed by immunoblotting (left) and RT-PCR (right), respectively. (B, C) RUNX2 promotes cell proliferation. U2OS cells were transfected as described in (A). Forty-eight hours after transfection, phase-contrast micrographs were taken. Scale bar = 100 μm (B). U2OS cells were transfected with 2.0 μg of the expression plasmid encoding RUNX2. At the indicated time points, the number of viable cells was measured (C).

Article Snippet: Twenty-four hours after treatment, cells were washed twice in ice-cold PBS, fixed in 3.7% folmaldehyde in PBS at room temperature for 30 min, permeabilized with 0.1% Triton X-100 in PBS at room temperature for 5 min and then blocked with 3% BSA in PBS at room temperature for 1 h. After blocking, cells were washed in ice-cold PBS and simultaneously incubated with mouse monoclonal anti-RUNX2 (8G5; Medical & Biological Laboratories, Nagoya, Japan) and rabbit polyclonal anti-p73 (Bethyl Laboratories, Montgomery, TX, USA) antibodies at room temperature for 1 h followed by the incubation with rhodamine-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG (Invitrogen) at room temperature for 1 h. After the extensive washing, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Peterborough, UK).

Techniques: Expressing, Transfection, Plasmid Preparation, Isolation, Western Blot, Reverse Transcription Polymerase Chain Reaction