Figure 9

Biochemical analysis of Nit1 aggregation. (A) Extracts from bromoxynil-treated plants were made 4% in Triton X100 and centrifuged for 10 min at 10,000 g and the pellet was imaged by confocal microscopy. (B) Western blot of protein extracts from leaf tissue of wild type and the Nit1-3 mutant showing that the antibody recognizes the Nit1 protein in wild type but not the null mutant. (C) Aggregation of Nit1 into the low speed pellet fraction during herbicide-induced cell death. Three week old seedlings were imbibed in solutions of bromoxynil (0, 125, 250, 500 μM) for 1 h then extracted, adjusted to 4% Triton X100 and centrifuged at 10,000 g for 10 min. Proteins in the supernatant and pellet were separated by SDS-PAGE and various proteins detected by western blotting. Antibodies against PIP2A (plasma membrane intrinsic protein) and cofillin were used to show complete separation of the two fractions.