Figure 2

Characterization of the NSD-1 protein. (A) Predicted NSD-1 polypeptide. The figure indicates the nonspliced form of NSD-1. Black and gray boxes indicate the transmembrane domain and the region corresponding to the intron in the spliced form of nsd-1, respectively. Green and blue boxes indicate the putative N-linked and O-linked glycosylation (mucin-like domain) sites, respectively. K (lysine) at 110 and G (glycine) at 118 indicate the amino acid residues derived from the resistant strain p50T, while E (glutamic acid) at 110 and R (arginine) at 118 are the amino acid residues derived from the susceptible strain J150. (B and (C) Western blot of recombinant NSD-1 in Sf9 cells using anti-His antibody (B) and anti-NSD-1 antibody (C). The membrane fraction was extracted from Sf9 cells and analyzed by western blot. The primary antibodies were anti-His antibody (1:3000) and anti-NSD-1 antibody (1:10,000), respectively. Lane 1, NSD-1-His-Res-nonspl-AcMNPV; 2, NSD-1-His-Sus-nonspl-AcMNPV; 3, Bac1-AcMNPV (negative control); 4, NSD-1-His-Res-spl-AcMNPV; 5, NSD-1-His-Sus-spl-AcMNPV. (D) Immunostaining of Sf9 cells infected with recombinant baculoviruses. The Sf9 cells were incubated with anti-NSD-1 antibody (1:100) followed by a secondary antibody labeled with AlexaFluor488 (1:500) (green) and counterstained with DAPI (1:1000) (red). DAPI staining (left); NSD-1 signals (center); merge (right). (E) Hypothetical membrane orientation of NSD-1 predicted by SOSUI (http://harrier.nagahama-i-bio.ac.jp/sosui/). The spliced form of NSD-1 lacks the gray region. The red filled circles indicate the positions of amino acids that vary between the resistant (p50T) and susceptible (J150) strains. The green and blue filled circles indicate the putative N-linked and O-linked glycosylation sites, respectively.