To quantitatively measure Yap/Tead signaling activity in vivo, HH5 avian embryos were co-electroporated with HOP-flash plasmid or its mutated version HIP flash (Addgene plasmid # 83467) (Kim and Gumbiner, 2015 (link)) and the pRL-TK control reporter vector (Promega, #E2231). The embryos were then cultured ex ovo until HH9. The two dorsal neural folds of each embryo were micro-dissected, and one explant was cultured in control media while the other was cultured in 2-DG containing media for 18h, in white, clear-bottom 96 well plates. Following explant culture, a luciferase assay was performed using the Dual-Luciferase Assay kit (Promega, #E1910) according to the manufacturer’s protocol. Post luminometer reading, firefly luciferase values were normalized to Renilla luciferase measurements to account for differences in transfection efficiency and number of cells. Normalized luciferase values were compared between control and 2-DG treated explants obtained from the same embryo.
Hip flash
Manufactured by Addgene
The HIP-flash is a lab equipment product. It is designed to perform high-intensity pulsed light (HIPL) treatment, a technique used in various applications such as surface modification and sterilization. The core function of the HIP-flash is to generate high-intensity, short-duration light pulses for specific experimental or processing requirements.
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Lab products found in correlation
Prl tk control reporter vector, by Promega (1 mentions)
Dual luciferase assay kit, by Promega (1 mentions)
Transit 2020 transfection reagent, by Mirus Bio (1 mentions)
Pgl4.74 hrluc tk, by Promega (1 mentions)
Pgl4.73 hrluc sv40, by Promega (1 mentions)
Hop flash plasmid, by Addgene (1 mentions)
Synergy 2 spectrophotometer, by Agilent Technologies (1 mentions)
Dual glo luciferase assay system, by Promega (1 mentions)
Hop flash, by Addgene (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Prl tk renilla plasmid, by Promega (1 mentions)
Dual luciferase reporter assay kit, by Promega (1 mentions)
4 protocols using hip flash
1
Quantifying Yap/Tead Signaling in Avian Embryos
2
TEAD-Luciferase Reporter Assay
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For TEAD-luciferase reporter assay, cells were co-transfected with either siControl or siRNF40, the HOP-flash luciferase reporter (Plasmid #83467, Addgene) or the HIP-flash (#83466, Addgene) and the pGL4.74[hRluc/TK] (E6921, Promega) Renilla control reporter using the TransIT-2020 transfection reagent (Mirus) based on manufacturer’s instructions. Finally, results were normalized to Renilla activity. For ATP-based luciferase activity, equal number of cells were subjected to ATP measurement using the ATP-based CellTiter-Glo Cell Viability Assay kit (Promega) based on manufacturer’s instructions. Results were plotted using GraphPad Prism v8.0.1. For a more detailed protocol, please refer to Supplementary Data.
3
Evaluating Hippo Pathway Activity
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For evaluation of Hippo-pathway activity, cells were seeded to 96-well white microplates and co-transfected with HOP-flash plasmid (#83467, Addgene) and pGL4.73 [hRluc/SV40] (Promega, Madison, WI, USA) plasmids using Lipofectamine 3000. For undifferentiated cells, assay was conducted 24 h after transfection. For differentiation experiments, myogenic differentiation was induced 24 h after transfection. Cell lysates preparation and substrate application was performed using the Dual-Glo Luciferase Assay System (Promega, USA). Luminescence was measured using Synergy2 spectrophotometer (BioTek, Winooski, VT, USA). Firefly luciferase activity was normalized to Renilla luciferase activity. As a negative control, cells were transfected with HIP-flash (#83466, Addgene) and hRLuc/SV40 plasmids.
4
Polaprezinc and ZnSO4 Modulation of Luciferase Activity
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Cells were seeded in triplicate in six-well culture plates at 1 × 105 cells and cultured for 24 h, and the luciferase reporter assay was performed as previously described53 . The cells were transfected with 500 ng of HOP-Flash (83467, Addgene) or HIP-Flash luciferase reporter plasmid (83466, Addgene) along with 10 ng of pRL-TK Renilla plasmid (Promega) using the Neon Transfection System (Invitrogen) according to the manufacturer’s instructions. The day after transfection, cells were treated with 50 μM polaprezinc or ZnSO4, respectively. Luciferase and Renilla signals were measured 48 h after transfection using a Dual Luciferase Reporter Assay Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions.
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