Nsp1_CYT interacts with ER at high resolution. cdc6-1 cells (PCCPL393) were arrested in G2/M phase for 3 h at 34°C, released for 3 h at RT, and then processed for immuno-EM. An anti-Nsp1 antibody followed by a 10 nm gold–conjugated secondary antibody were used to localize Nsp1. (A) Plot of the percentage of gold particles from a single experiment both at (left; n = 71) and outside (right; n = 46) the nucleus in association with the indicated cellular structures. (B and C) EM micrographs of a mother and bud (daughter) of the same cell. N, nucleus; V, vacuole. Asterisks indicate NPCs and arrowheads point to gold particles. Bars, 1 µm. (D–F) High-magnification views of gold particles in association with internal ER (D) and cortical ER (E and F). Arrows delimit the two ER membranes with intervening cistern. Arrowheads point to gold particles. PM, plasma membrane. Bars, 100 nm.