Figure 1.

Identification of the NAG protein as a component of the syntaxin 18 complex. (A) Triton X-100 extracts of 293T cells were immunoprecipitated with an anti-p31 antibody (mAb 5C3) attached to protein G-Sepharose 4B. The coprecipitated proteins were resolved by SDS-PAGE, stained with silver, and analyzed by LC-MS/MS. An asterisk denotes immunoglobulin heavy chain. (B) Triton X-100 extracts of 293T cells were incubated at 16°C for 60 min with 10 μg/ml NSF and 5 μg/ml α-SNAP in the presence or absence of 8 mM Mg²⁺ and 0.5 mM ATP. After incubation, the samples were immunoprecipitated with a mAb against p31 (lanes 2 and 3) or syntaxin 18 (lanes 5 and 6). The precipitated proteins were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. (C) Cell homogenates were subjected to Nycoprep density gradient centrifugation and analyzed by immunoblotting with the indicated antibodies. (D) HeLa cells were transfected with the plasmid encoding FLAG-NAG. At 24 h after transfection, the cells were left untreated (top row; − Dig) or treated with digitonin (bottom row; + Dig), and double-stained with antibodies against FLAG and Bap31 (left) or Sec61β (right). Bar, 20 μm.