Fig. 1

A specific FMNL2 isoform is over-represented in invasive cells. a RT-PCR was performed on total RNA harvested from SW480, SW620 and A375 cells. Specific primers were used to demonstrate the presence of four alternatively spliced FMNL2 isoforms identified in an initial RT-PCR analysis (see Results). b The four alternatively spliced mRNA are predicted to encode proteins differing only at amino acid residues C-terminal to the DAD domain. c qPCR was used to assess the levels of expression of each isoform in comparison to total FMNL2 in the indicated colorectal and melanoma cell-lines as well as in non-transformed primary endothelial cells and melanocytes. The TQS isoform was predominant in all the invasive cell-lines that were assessed. We were unable to obtain reliable qPCR data for the PMR isoform. d Isoform specific antibodies were raised against peptides corresponding to the unique domain of each C-terminal tail. Full-length derivatives of the indicated FMNL2 isoforms bearing N-terminal myc epitope tags were expressed in U2OS cells by transient transfection and the resulting cell lysates were immunoblotted with the indicated antibodies. Total FMNL2 protein was detected using an antibody directed against FMNL2 codons 599–1045. Equivalent samples were loaded on a separate gel and probed with α-myc antibodies. The α-FMNL2 blot was stripped and re-probed with α-TQS antibody; the α-myc blot was stripped and re-probed with α-PMR antibody. Equivalent samples were loaded on separate gels to detect ITM and YHY e Immunoblot analysis using antibodies directed against the C-terminal tails of each of the predicted FMNL2 isoforms confirms the qPCR analysis. Total FMNL2 protein was detected using pan FMNL2 antibody shown in 1D. Isoform specific antibodies were raised against peptides corresponding to the unique domain of each C-terminal tail. Equivalent samples were loaded on separate gels to eliminate concerns regarding incomplete stripping. The pan-FMNL2 blot was stripped and re-probed with anti α-tubulin for a loading control