Genetic Manipulation of Cellular Pathways

On day 1, cells were seeded into Nunclon^TM Delta Surface plates (Nunc, Roskilde, Denmark), and transfected twice, on days 2 and 3, serum-free with either 25 or 50 nM of prevalidated siRNAs specific for RAC1B [6 (link)], SMAD2, SMAD3 (both from Dharmacon, Lafayette, CO, USA), BGN [7 (link)], or the respective scrambled controls, and/or pcDNA3-based expression vectors for SMAD3, SMAD3-mut3A, or SMAD2, for 4 h using Lipofectamine 2000 (Life Technologies). Additional validation of these siRNAs was performed for RAC1B [6 (link)] and SMAD2/SMAD3 (this study).

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Otterbein H., Lehnert H, & Ungefroren H. (2019). Negative Control of Cell Migration by Rac1b in Highly Metastatic Pancreatic Cancer Cells Is Mediated by Sequential Induction of Nonactivated Smad3 and Biglycan. Cancers, 11(12), 1959.

Publication 2019

NunclonTM Delta Surface plates SMAD3 Lipofectamine 2000

Cells Lipofectamine 2000 Serum Sirnas Smad2 Smad3 Vectors

Corresponding Organization : University of Lübeck

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Protocol cited in 2 other protocols

Variable analysis

independent variables

Transfection with 25 or 50 nM of prevalidated siRNAs specific for RAC1B, SMAD2, SMAD3, or BGN
Transfection with pcDNA3-based expression vectors for SMAD3, SMAD3-mut3A, or SMAD2

dependent variables

Not explicitly mentioned

control variables

Seeded cells into Nunclon™ Delta Surface plates
Transfection with respective scrambled controls

controls

Positive control: Respective scrambled controls for siRNA transfections
Additional validation of siRNAs for RAC1B and SMAD2/SMAD3

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