Fig. S3

fak MO efficacy and specificity. (A) RT-PCR for control MO and fak MO injected embryos. Whole tissue lysate was analyzed. fak MO injection resulted in 2 abnormal mRNA products (arrows 1 and 2). EF1 was used as an RT-PCR control. Primers used for RT-PCR: fak exon 11 forward 5-CACCTTGCCAACTTCACTCA-3; fak exon 22 reverse 5- GTGAATCGTGGGCGTTTACT-3; EF1 forward, 5-GATGCACCACGAGTCTCTGA-3; and EF1 reverse, 5-TGATGACCTGAGCGTTGAAG-3. fak RT-PCR products were cloned into pGEM using the pGEM T-Easy Vector System Kit (Promega) and sequenced. (B) Sequence analysis of the two RT-PCR product variants from (A) resulted in the detection of two truncated mRNA amplicons caused by partial or complete exon deletion, each resulting in early stop codons as indicated in the diagram. Both truncations eliminate the autophosphorylation site Y397. (C) Western Blot analysis of fak morphant lysate demonstrating downregulation of full length Fak (asterisks ** at 125 kDa). Injected embryos were manually dechorionated and deyolked. Proteins were analyzed on 8% SDS-PAGE gels. Blots were blocked in 4% non-fat milk or 5% BSA and probed with antibody in 3% BSA. FAK C-20 (1:1000 dilution; sc-558) and FAK (1:200 dilution; Invitrogen AHO0502). GAPDH was used as a loading control (1:25,000 dilution, ab22555). Blots were visualized using Enhanced Chemiluminscence. (D) Representative brightfield images and quantification of fak morphant embryo rescue experiments. Control MO or fak MO was co-injected with mGFP, wild-type human FAK, or human FAKY397E mRNA. Each condition had equal total amounts of mRNA injected. Embryos were analyzed at prim-6 for MHB defects. Each embryo was scored as having a normal, mild, or severe phenotype. Mild phenotypes are represented in D. Control MO + mGFP (n=38), control MO + wt hFAK (n=42), fak MO + mGFP (n=55), fak MO + hFAK (n=63), control MO + hFAKY397E (n=51), fak MO + hFAKY397E (n=54). Anterior is to the left in all images. Arrowheads indicate MHBC.