mouse anti human p21 antibody  (Bio-Rad)

Journal: Antioxidants

Article Title: Exploring the Anticancer Potential of the Multistrain Probiotic Formulation OxxySlab in Bladder Cancer Cell Lines

doi: 10.3390/antiox14111282

Figure Lengend Snippet: Senescence induction in BC cells following OxxySlab lysate treatment. Cell lines were treated with OxxySlab lysate (75–150 μg/mL) for 48 h. Senescent cells were identified by β-galactosidase staining (β-gal). Representative phase-contrast images of T24 ( A ), 5637 ( E ) and SV-HUC-1 ( I ) β-gal-positive cells (blue staining) are shown. Quantification of senescence was performed by measuring the Mean Gray Value of blue staining intensity using ImageJ software, with data expressed as Inverted Mean Gray Value (255 − MGV), which directly reflects β-gal activity. Data are expressed as mean ± SEM of two independent experiments in triplicate. Western blot analysis of the senescence markers p21, p53 and p16 was performed in untreated (CNTR) and OxxySlab -treated T24 ( B – D ), 5637 ( F – H ), and SV-HUC1 ( J – L ) cells. Protein levels were quantified by densitometry, normalized to GAPDH, and expressed as fold change relative to CNTR of three independent experiments (mean ± SEM). Statistical significance was assessed by one-way ANOVA followed by Tukey post hoc test (* p < 0.05, ** p < 0.01).

Article Snippet: Following incubation with 5% non-fat dry milk in Tris-buffered saline for 1 h at room temperature, the membranes were incubated overnight at 4 °C with primary antibodies: goat anti-human vimentin polyclonal antibody (Chemicon International, Temecula, CA, USA; dilution 1:100), mouse anti-human E-cadherin monoclonal antibody (Cell Signaling Technology; dilution 1:1000), rabbit anti-human β-catenin monoclonal antibody (Cell Signaling Technology; dilution 1:1000), rabbit anti-human p21 polyclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA dilution 1:1000); rabbit anti-human phospho-Nrf2 monoclonal antibody (S40) (1:2000, Abcam, Cambridge, UK); rabbit anti-human p16 polyclonal antibody (Santa Cruz Biotechnology, dilution 1:200); mouse anti-human p53 monoclonal antibody (Santa Cruz Biotechnology, dilution 1:1000); mouse anti-human GAPDH monoclonal antibody (Immunological Sciences, Rome, Italy; dilution 1:1000).

Techniques: Staining, Software, Activity Assay, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: HDBECs (2×10 5 ) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2-AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre-incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre-incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1×10 5 cells). Total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein, (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; PAR2, protease-activated receptor 2.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Incubation, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Immortalised human epithelial cells (human telomerase reverse transcriptase-human pancreatic nestin-expressing ductal cells) were cultured in 25 cm 2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated wks, total RNA was isolated from one group and the mRNA quantified by RT-qPCR, against β-actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 mRNA (n=3), and (B) the relative amounts of p21 CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21 CIP1/WAF1 protein (using a mouse anti-human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Reverse Transcription, Expressing, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Western Blot

Journal: Molecular Medicine Reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16 INKa , CDK interacting protein/Wildtype p53-activated fragment p21 CIP1/WAF1 and Alternative reading frame p14 ARF , which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21 CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16 INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16 INKa . TF, tissue factor; PAR2, protease-activated receptor 2, Rb, retinoblastoma protein; E2F, early region 2 binding factor; p21 CIP1/WAF1 , CDK interacting protein/wildtype p53-activated fragment; p16 INK4a , inhibitor of CDK; p14 ARF , alternative reading frame.

Article Snippet: The membranes were probed overnight at 4°C with either a goat anti-human p16 antibody (1:2,000 v/v; cat. no. AF5779; R&D Systems Europe, Ltd.), a mouse anti-human p21 antibody (WA-1; cat. no. MCA2325; Bio-Rad Laboratories, Inc.), a rabbit anti-human p14 antibody (cat. no. abx013162; Abbexa, Ltd.) or a rabbit anti-human Cyclin E1 antibody (cat. no. abx012757; Abbexa, Ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay

Journal: Molecular medicine reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation.

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Figure 2. HDBECs (2x105) were incubated for 24 h with recombinant TF (0, 0.5 and 2 U/ml) or PAR2‑AP (SLIGKV; 20 µM), or with recombinant TF (0.5 U/ml) that was pre‑incubated for 60 min with 10H10 or HTF1 antibodies (20 µg/ml). Groups of cells were also pre‑incubated for 60 min with AIIB2 or SAM11 antibodies (20 µg/ml), prior to the addition of TF. The cells were harvested after 24 h, into separate aliquots (1x105 cells). Total RNA was isolated from one group and the mRNA quantified by RT‑qPCR, against β‑actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53‑activated fragment p21CIP1/WAF1 mRNA (n=5), and (B) the relative amounts of p21CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21CIP1/WAF1 protein, (using a mouse anti‑human antibody) and against GAPDH. TF, tissue factor; PAR2, protease‑activated receptor 2.

Article Snippet: The membranes were probed overnight at 4 ̊C with either a goat anti‐human p16 antibody (1:2,000 v/v; cat. no. aF5779; r&d Systems europe, ltd.), a mouse anti‐human p21 antibody (Wa‐1; cat. no. Mca2325; Bio‐rad laboratories, inc.), a rabbit anti‐human p14 antibody (cat. no. abx013162; abbexa, ltd.) or a rabbit anti‐human cyclin e1 antibody (cat. no. abx012757; abbexa, ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Incubation, Recombinant, Isolation, Western Blot

Journal: Molecular medicine reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation.

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Figure 5. Immortalised human epithelial cells (human telomerase reverse transcriptase‑human pancreatic nestin‑expressing ductal cells) were cultured in 25 cm2 flasks and supplemented with recombinant TF (0.5 U/ml) or were untreated. The cells were harvested on the indicated wks, total RNA was isolated from one group and the mRNA quantified by RT‑qPCR, against β‑actin. Other aliquots were analysed by western blotting. The data show the amounts of (A) CDK interacting protein/Wildtype p53‑activated fragment p21CIP1/WAF1 mRNA (n=3), and (B) the relative amounts of p21CIP1/WAF1 protein (n=3) calculated from (C) the western blots of p21CIP1/WAF1 protein (using a mouse anti‑human antibody) and against GAPDH. TF, tissue factor; Wk, week.

Article Snippet: The membranes were probed overnight at 4 ̊C with either a goat anti‐human p16 antibody (1:2,000 v/v; cat. no. aF5779; r&d Systems europe, ltd.), a mouse anti‐human p21 antibody (Wa‐1; cat. no. Mca2325; Bio‐rad laboratories, inc.), a rabbit anti‐human p14 antibody (cat. no. abx013162; abbexa, ltd.) or a rabbit anti‐human cyclin e1 antibody (cat. no. abx012757; abbexa, ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Cell Culture, Recombinant, Isolation, Western Blot

Journal: Molecular medicine reports

Article Title: Tissue factor signalling modifies the expression and regulation of G1/S checkpoint regulators: Implications during injury and prolonged inflammation.

doi: 10.3892/mmr.2024.13404

Figure Lengend Snippet: Figure 9. Proposed model for the mechanism by which the level of TF on the cell surface may have differential outcomes on G1/S checkpoint regulation. The presence of TF on the cell surface differentially upregulates the expression of Inhibitor of CDK p16INKa, CDK interacting protein/Wildtype p53‑activated fragment p21CIP1/WAF1 and Alternative reading frame p14ARF, which is dependent on the concentration of TF and the ability of the cell to dissipate excess TF. Therefore, alterations in p21CIP1/WAF1 are highly effective in the regulation of the cellular response to acute stress. The interplay between these proteins modulates the signal permitting passage through the cell cycle, or alternatively its arrest. Consequently, the concentration of TF may be an ideal gauge for determining the level of cellular damage. However, the adaptive loss of p16INK4a function may be promoted by prolonged inflammation leading to permissive transition through the G1/S checkpoint, even in the absence of mutational loss of p16INKa. TF, tissue factor; PAR2, protease‑activated receptor 2, Rb, retino‑ blastoma protein; E2F, early region 2 binding factor; p21CIP1/WAF1, CDK interacting protein/wildtype p53‑activated fragment; p16INK4a, inhibitor of CDK; p14ARF, alternative reading frame.

Article Snippet: The membranes were probed overnight at 4 ̊C with either a goat anti‐human p16 antibody (1:2,000 v/v; cat. no. aF5779; r&d Systems europe, ltd.), a mouse anti‐human p21 antibody (Wa‐1; cat. no. Mca2325; Bio‐rad laboratories, inc.), a rabbit anti‐human p14 antibody (cat. no. abx013162; abbexa, ltd.) or a rabbit anti‐human cyclin e1 antibody (cat. no. abx012757; abbexa, ltd.), each diluted 1:3,000 (v/v) in TBST.

Techniques: Expressing, Concentration Assay, Binding Assay