mouse anti human p21 antibody  (Bio-Rad)

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

Journal: Antioxidants

Article Title: The microRNA Let-7f Induces Senescence and Exacerbates Oxidative Stress in Retinal Pigment Epithelial Cells

doi: 10.3390/antiox13060646

Figure Lengend Snippet: Let-7f induces a senescence phenotype in human retinal pigment epithelial cells. ARPE-19 cells were transfected with 50 nM let-7f mimic for 48 h and senescence was examined through various assays. ( A ) Proliferating cells were identified via immunofluorescent staining of Ki67 (green) positive cells and nuclei (DAPI, blue) (10×). ( B ) The percent of Ki67 positive cells was calculated based on the number of Ki67 positive cells divided by the total number of cells (DAPI positive) and expressed versus scramble control (Sc). ( C ) Representative images of senescence-associated β-galactosidase (SA-β-gal) staining used to detect senescent ARPE-19 cells. Senescent cells were identified by the development of blue coloration and an enlarged, flattened morphology as indicated by the red arrows (10×). ( D ) Percentage of senescent ARPE-19 cells was compared to scramble control (Sc). ( E ) The protein expression levels of cyclin-dependent kinase inhibitors p16 INK4a and p21 Waf/Cip1 were detected via western blotting. Band densitometry was calculated using ImageLab Software and normalized to β-actin. Results were presented as a fold change compared to scramble control cells (Sc). * p < 0.05, *** p < 0.001 vs. Sc. ns: not significant. All data are expressed as the mean ± SD ( n = 3).

Article Snippet: Membranes were blocked in 5% BSA for 2 h and incubated with mouse anti-human p16 INK4a (1:500, Lifespan Biosciences, Shirley, MA, USA), mouse anti-human p21 Waf/Cip1 (1:500, Lifespan Biosciences) or rabbit anti-human β-actin (1:5000, Novus Biologicals, Centennial, CO, USA) at 4 °C overnight.

Techniques: Transfection, Staining, Control, Expressing, Western Blot, Software