(A) The six possible topological orientations for Src1-L and the two for Src1-S are shown

N, N terminus; C, C terminus; INM, inner nuclear membrane; ONM, outer nuclear membrane. Experimentally determined topologies are boxed in red. (B) Schematic illustration of the TEV-based method to determine the topology of Src1 membrane insertion (left). Under inducing conditions, the TEV protease is expressed and cleaves at TEV cleavage sites when accessible at the nuclear side. TEV-CS, TEV cleavage site. Cells expressing N- (TAP-Src1) or C-terminal TAP-tagged Src1 (Src1-L–TAP and Src1-S–TAP) or Δ cells containing C-terminal TAP-tagged Src1-L–ΔM2 were grown either under noninducing (−) or inducing (+) conditions (right). Whole cell extracts were analyzed by Western blotting using anti-ProtA, anti-myc (TEV protease), and anti-Arc1 antibodies (loading control).Copyright information:Taken from "The inner nuclear membrane protein Src1 associates with subtelomeric genes and alters their regulated gene expression"The Journal of Cell Biology 2008;182(5):897-910.Published online 8 Sep 2008PMCID:PMC2528585.