Retroviral expression vector (pBabe-puro) encoding Syndecan-4 fused to an extracellular HA-tag was kindly provided by Dr. Mark Morgan (University of Liverpool, UK) [36 ]. Syndecan-4-HA fragment was subcloned into EcoRI/XhoI sites of pcDNA3.1(+) plasmid. For the live cell experiments we used mCherry-vinculin (modified pEGFP-C1-mCherry plasmid harboring a deletion of the GFP gene sequence), kindly provided by Dr. Vicente Torres (Universidad de Chile, Chile) [37 ]. pEGFP-C1-Tiam1-PHnCCEx containing a mutation in the RhoK phosphorylation site encoding for a dominant negative mutant of Tiam1 was kindly provided by Dr. María Paz Marzolo (P. Universidad Católica de Chile, Chile) [38 ].
To downregulate PAR-3 expression we used siRNA ON-TARGET plus SMART pool (Thermo Scientific) against rat PAR-3 or FlexiTube siRNA Mm_Pard3_4 (Qiagen) targeting mouse PAR-3. A mix of two Tiam1 siRNA targeting the human Tiam1 with 88% homology with the rat Tiam1 sequence (Dharmacon) was used to lower Tiam1 expression. Silencer® Negative Control No. 1 (Ambion™) or AllStars (Qiagen) were used as siRNA negative control. Plasmids and siRNAs transfections of DI TNC1 cells were performed with Amaxa Nucleofector following manufacturer’s recommendations for astrocytes (Amaxa Biosystems, Lonza). Lipofectamine RNAiMAX Reagent and Lipofectamine 3000 (Thermo Fisher Scientific) were used to transfect MEFs.
To downregulate PAR-3 expression we used siRNA ON-TARGET plus SMART pool (Thermo Scientific) against rat PAR-3 or FlexiTube siRNA Mm_Pard3_4 (Qiagen) targeting mouse PAR-3. A mix of two Tiam1 siRNA targeting the human Tiam1 with 88% homology with the rat Tiam1 sequence (Dharmacon) was used to lower Tiam1 expression. Silencer® Negative Control No. 1 (Ambion™) or AllStars (Qiagen) were used as siRNA negative control. Plasmids and siRNAs transfections of DI TNC1 cells were performed with Amaxa Nucleofector following manufacturer’s recommendations for astrocytes (Amaxa Biosystems, Lonza). Lipofectamine RNAiMAX Reagent and Lipofectamine 3000 (Thermo Fisher Scientific) were used to transfect MEFs.