Ki 67 ki67

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Ki-67 (Ki67) is a protein that is associated with cellular proliferation. It is commonly used as a marker to determine the growth fraction of a cell population.

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3 protocols using ki 67 ki67

Quantification of Cellular Proliferation

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HRVECs were fixed with 4% paraformaldehyde, blocked with 3% FBS albumin (Sigma-Aldrich), and next incubated with polyclonal rabbit anti-proliferating antigen KI67 (Ki67) (Santa Cruz Biotechnology, CA, USA) primary antibody (1:200) overnight at 4°C. Secondary antibody Alexa Fluor 488 donkey anti-rabbit (1:200) was employed for 1 h at 37°C. Nuclei were visualized by staining with 4′, 6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich). An Olympus FV1000 confocal laser scanning microscope (Olympus, Japan; magnification, x200) was used to acquire images.

Li W, & Xiao H. (2021). Scutellaria barbata D. Don Polysaccharides Inhibit High Glucose-Induced Proliferation and Angiogenesis of Retinal Vascular Endothelial Cells. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 14, 2431-2440.

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Detailed Protocol for FOXP3 and TGF-β Signaling

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The primers used in this study were listed in Supplementary Table S6. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-flag-Smad2 (Addgen plasmid #14042) and CS2-flag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), p53 (DO-1, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Jia H., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B, & Chen G.G. (2020). Nuclear FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc. Oncogenesis, 9(10), 97.

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Molecular Pathways in Transcriptional Regulation

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The primers used in this study were listed in Supplementary Table S1. The pcDNA3.1-FOXP3, pcDNA3.1, Flag-pcDNA3.1-FOXP3, Flag-pcDNA3.1 were produced in our Lab. CS2-ag-Smad2 (Addgen plasmid #14042) and CS2-ag-Smad3 (Addgen plasmid #14052) was a gift from Joan Massague, pcDNA3-Flag-Smad4 (Addgen plasmid #80888) was a gift from Aristidis Moustakas, Pbv-Luc (Addgen plasmid #16539) and c-Myc promoter del-1 (Addgen plasmid #16601) was a gift from Bert Vogelstein. SiRNA-FOXP3 (sc-43569) and siRNA-Control (sc-43569) were from Santa Cruz Biotechnology. The primary antibodies for western blot were as follows: FOXP3 (150D/E4, Invitrogen, 1:1000), FOXP3Δ3 (16J4G6, Novus,1:1000), PCNA (PC10, Santa Cruz, 1:2000), Bax (B-9, Santa Cruz, 1:1000), Bcl2 (C-2, Santa Cruz, 1:1000), Smad2 (A-11, Santa Cruz, 1:1000), Smad3 (38-Q, Santa Cruz, 1:1000), Smad2/3 (A-3, Santa Cruz, 1:1000), p-Smad2(s467, Abcam, 1:1000), p-Smad3(1D9, Santa Cruz, 1:1000), Smad4 (B-8, Santa Cruz, 1:1000), c-Myc (9E10, Santa Cruz, 1:1000), and GAPDH (6C5, Santa Cruz, 1:2000). The primary antibodies for immunohistochemistry were listed below: FOXP3 (150D/E4, Invitrogen, 1:100), FOXP3Δ3 (16J4G6, Novus,1:100), Ki-67 (Ki67, Santa Cruz, 1:50), c-Myc (9E10, Santa Cruz, 1:100), Smad2/3 (A-3, Santa Cruz, 1:100), and p-Smad3 (Santa Cruz, 1:100).

Gong Z., Hao J., Yu J., Liu Y., Ren J., Yang S., Hu B., Liu L., Lai P.B., & Chen G.G. (2020). FOXP3 inhibits tumor growth and induced apoptosis in hepatocellular carcinoma by targeting c-Myc.

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