Luciferase assay was measured using Dual-Luciferase Reporter assay kit (Promega). Cultured cells were lysed with passive lysis buffer for 15 min at room temperature. Renilla (Rluc) and Firefly (Fluc) enzymatic activities were assayed with the substrates supplied with the kit using Centro XS3 LB960 machine (Berthold technologies). Typically, expression of Fluc was used to normalize the Rluc expression. Myc activity was measured using Myc-reporter constructs (pGL3-M4 and pGL3-M4-mut, (Nagel et al., 2008 (link)). MCF7 cells (1x105 cells) were plated in 12-well plates and transfected with 200ng of the reporter constructs using Fugene-6 (Promega). Luciferase activity was measured 36 hr after transfection.
Centro xs3 lb960 machine
Manufactured by Berthold Technologies
Sourced in Germany
The Centro XS3 LB960 is a compact and versatile laboratory machine designed for various analytical applications. It features high-precision measurement capabilities and can perform tasks such as sample preparation, liquid handling, and detection. The machine's core function is to provide reliable and consistent results for laboratory researchers and technicians.
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4 protocols using centro xs3 lb960 machine
1
Luciferase Assay for Myc Activity
2
Enhancer-Driven Luciferase Assay for YAP/TRAM2 Regulation
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The enhancer E region was PCR amplified from DNA of MCF10A cells and cloned into downstream of Poly-A of the firefly luciferase reporter gene in the pGL3-promoter (Promega) vector. For transfection of these plasmids, 1 × 105 of MCF10A-YAP5SA, MCF10A-Control, MCF10A-TRAM2 cells were seeded in 6-well plates. The next day, 200 ng of each construct (pGL3-promoter, pGL3-promoter-enhancer/forward, and pGL3-promoter-enhancer/reverse) were co-transfected with 20 ng of Renilla luciferase reporter construct using Fugene-6 (Promega) following the manufacturer’s protocol. Luciferase activity was measured 24 h post-transfection using the dual-luciferase reporter assay kit (Promega). Cells were lysed directly on the plate with passive lysis buffer for 15 min at room temperature. Firefly and Renilla luciferase activity was measured using a Centro XS3 LB960 machine (Berthold technologies).
3
YAP1 Transcriptional Activity Assay
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For assessment of YAP1 activity, 1 × 105 cells were seeded in 6-well plates at least in triplicates, co-transfected on the following day with 200 ng of a YAP1 luciferase reporter described previously [60 (link)] and 20 ng Renilla as transfection control using 1 µg Polyethyleneimine “MAX” (Polysciences, Warrington, PA, United States) per µg DNA. Luciferase activity was measured 24 h post-transfection using the Dual-Luciferase Reporter assay kit (Promega, Madison, WI, United States) according to the manufacturer’s protocol and a Centro XS3 LB960 machine (Berthold Technologies, Bad Wildbad, Germany) or an Infinite M200 PRO (Tecan, Männedorf, Switzerland). Luciferase activity was normalized to Renilla. Each experiment was performed at least twice on different days.
4
Enhancer-mediated Luciferase Assay
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The constructs with the enhancers were cloned based on pGL3-promoter (Promega) vector. The enhancer region was PCR amplified from BJ genomic DNA and inserted downstream of the firefly luciferase reporter gene. The transfection was performed by seeding 1 × 105 of cultured cells in six-well plates. The next day, 500 ng of each construct (pGL3-promoter, pGL3-EnhAP1-OIS1-Fw, and pGL3-EnhAP1-OIS1-Rv) were co-transfected with 50 ng of Renilla luciferase reporter construct using Fugene-6 (Promega) following the manufacturer’s protocol. Luciferase reporter assay was performed 24 h after transfection using a Dual-Luciferase Reporter assay kit (Promega). Cells were lysed directly on the plate with passive lysis buffer for 15 min at room temperature. Firefly and Renilla luciferase activity were measured with the substrates from the kit using Centro XS3 LB960 machine (Berthold Technologies). For BJ-indRASG12V, cells were pre-treated with 100 nM 4-OHT for 48 h before transfection. For HCT116, cells were treated with UV-C (50 J/m2) or MG132 (5 μM) 18 h after transfection. The luciferase assay was performed 5 h after treatment.
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