Affinityscript kit

For both mESCs and for zebrafish embryos cDNA was generated with the AffinityScript kit (Agilent) as previously described^{34 (link)}. Briefly, 500 ng of RNA were retrotranscribed using random primers. For qPCR, the cDNA was diluted 1:10. All qPCRs were performed with the PowerUp SYBR Green Master Mix (ThermoFisher Scientific) with 300 nM of each primer and 2 μl of diluted cDNA. Fluorescence acquisition was performed on either a QuantStudio 5 System machine or QuantStudio 12 Flex (ThermoFisher Scientific). Primers are listed in Supplementary Table 1. For the zebrafish samples, quantification for relative gene expression was performed using the comparative Ct method and target gene expression was normalized to actin. In addition, for most of the qPCRs, time course values for MZsmad4a and Msmad4a mutants were normalized to the WT expression levels at 75% epiboly for each replicate. For SB-505124 dose responses, WT and MZsmad4a mutant values were normalized to each DMSO treatment value. Instead, for mESCs experiments, target gene expression was normalized to Gapdh levels. Fold changes in gene expression between WT cells and SMAD4 knockout clones were obtained by normalizing expression values to the 2-h WT sample in the case of the BMP4 inductions and to the 8-h WT sample in the case of the Activin A inductions.

Full-length vinculin cDNA was isolated from a human A431 cell line by extracting mRNAs using the RNeasy Kit (QIAGEN), followed by a reverse transcriptase reaction with a specific primer (5’-GGTGCCTACTGGTACCAGGGAG-3’) using the AffinityScript kit (Agilent). The PCR product (using the forward 5’-ATATATGCTCTTCTAGTATGCCAGTGTTTC-3’, and the reverse 5’-TATATAGCTCTTCATGCCTGGTACCAGGG-3’ primers) of 1066 amino acids long, was cloned into a pFBXC3GH insect cell expression vector containing a C-terminal 3C protease cleavage site followed by a GFP and His tag, using the fragment exchange (FX) cloning strategy (Geertsma and Dutzler, 2011 (link)). Protein expression was performed by generating a recombinant baculovirus for Sf9 insect cell infection at a density of 2.0 × 10⁶ mL⁻¹ using the Bac-to-Bac system (Invitrogen). Briefly, the recombinant vinculin-GFP encoding plasmid pFBMS was transformed into DH10Bac E. coli cells, which enabled the transposition of the recombinant gene into the bacmid genome. The recombinant bacmid DNA was then isolated and transfected into Sf9 cells to generate the full-length vinculin-expressing baculovirus.