Figure 1: Full-length PB1-F2 variants associate with mitochondria.

(a) Cellular fractions from A/PR8 (16 HA U ml⁻¹)-infected HEK293 cells were collected by differential centrifugation and analyzed by Western blotting using the indicated organelle markers. C, cytosolic fraction; H, heavy membrane fraction; L, light microsomal membrane fraction. (b) Subcellular localization of PB1-F2 variants. The indicated constructs (PR8, 88W, 58W and 12S) were expressed in HeLa cells, and immunofluorescence with PB1-F2 specific antibodies was used to detect expression (middle panels). Mitochondria in the same cells were identified by stable expression of mitochondrially targeted red fluorescent protein (Mito-RFP, left panels). The bottom four rows depict other organelle markers for the endoplasmic reticulum (ER-green and anti-PDI) and peroxisomes (anti-catalase). Scale bar, 10 μm. (c) Each full-length PB1-F2 variant was synthesized in vitro and incubated with isolated mitochondria (Mt) from HEK293 cells for the indicated times at 25 °C. The reactants were then treated with proteinase K and analyzed by Western blotting. Bottom panel, CoxIV was used as a loading control.