Experiments were performed in HeLa Kyoto cells unless otherwise stated. Knockdowns were performed using Lipofectamine 2000 as per manufacturer’s instructions with double transfections of 48 h. Exogenously expressed constructs were transfected using Lipofectamine 2000 for 24 h before the assay. Mutants were cloned using GeneString technology. TRAILR4 3’UTR was cloned from Origene clone SC117708 into psiCHECK2 using Gene String technology. TRAILR4 3’UTR fragments were cloned by PCR amplification of the indicated regions and cloned into psiCHECK2. Renilla and Firefly luminescence were measured 48 h post transfection. Crosslinking followed by immunoprecipitation was performed as previously described41 . To assess cell sensitivity to TRAIL-mediated apoptosis, cells were treated with TRAIL for 24 h, and cell viability was assayed by MTT assay (Millipore) or by Annexin V/PI flow cytometry (Biolegend) as per manufacturer’s instructions. Standard Western Blotting techniques were used, and full scans of important blots along with protein ladder markers are shown in Supplementary Fig. 9 .
Annexin 5 pi flow cytometry
Manufactured by BioLegend
Annexin V/PI flow cytometry is a technique used to detect and quantify apoptosis (programmed cell death) in cells. Annexin V is a protein that binds to phosphatidylserine, a phospholipid that is exposed on the surface of cells undergoing apoptosis. Propidium iodide (PI) is a fluorescent dye that can only enter cells with compromised cell membranes, typically found in late apoptotic or necrotic cells. By using a combination of Annexin V and PI, flow cytometry can distinguish between viable, early apoptotic, late apoptotic, and necrotic cell populations.
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2 protocols using annexin 5 pi flow cytometry
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TRAIL-mediated apoptosis regulation by TRAILR4
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TRAIL-mediated apoptosis regulation by TRAILR4
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Experiments were performed in HeLa Kyoto cells unless otherwise stated. Knockdowns were performed using Lipofectamine 2000 as per manufacturer’s instructions with double transfections of 48 h. Exogenously expressed constructs were transfected using Lipofectamine 2000 for 24 h before the assay. Mutants were cloned using GeneString technology. TRAILR4 3’UTR was cloned from Origene clone SC117708 into psiCHECK2 using Gene String technology. TRAILR4 3’UTR fragments were cloned by PCR amplification of the indicated regions and cloned into psiCHECK2. Renilla and Firefly luminescence were measured 48 h post transfection. Crosslinking followed by immunoprecipitation was performed as previously described41 . To assess cell sensitivity to TRAIL-mediated apoptosis, cells were treated with TRAIL for 24 h, and cell viability was assayed by MTT assay (Millipore) or by Annexin V/PI flow cytometry (Biolegend) as per manufacturer’s instructions. Standard Western Blotting techniques were used, and full scans of important blots along with protein ladder markers are shown in Supplementary Fig. 9 .
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