Figure 2.
Yif1B mRNA and protein expression in rat tissues and direct interaction of Yif1B from rat brain homogenates with the 5-HT1AR C-tail. A, Yif1B mRNA expression in rat tissues was analyzed by Northern blot. Each lane represents 3 μg of mRNA from each rat tissue; the human lung is used as a control for the hybridization specificity. The 32P-labeled Yif1B probe consisted of 425 bp in the 5′end of the rat cDNA. B, C, Yif1B protein expression in LLC-PK1 cells untransfected or transfected with Yif1B (B) and in rat tissues (C) was analyzed by Western blot using anti-Yif1B affinity-purified polyclonal antibody (1:1000). Each lane represents 1 μg of total protein, and the same Western blot was also revealed with actin antibody at the bottom. D–G, GST pull-down experiments on Yif1B-transfected LLC-PK1 cells (D) and brain tissue homogenates: cerebellum (E), hippocampus (F), or raphe (G). Extracts were incubated with beads coupled to GST alone or to GST fused with CT1A, I31A, or CT1B. Interaction was analyzed by Western blot with anti-Yif1B affinity-purified polyclonal antibody (1:1000). “Input” represents 2.5 μg of proteins (tissue extract prepared as in C). Results are representative of at least three independent experiments.