Figure 5.

p180 depletion perturbs ascorbate-stimulated de novo biosynthesis in polysome fractions on the ER membrane. The de novo biosynthesis activity was estimated using a non-RI labeling system with AHA in HEL cells. The cells were cultured in the presence or absence of ascorbate and treated with a control or p180-specific siRNA. After incubation with AHA, the membrane fractions were fractionated on a 15–50% sucrose gradient. The AHA-incorporating proteins were labeled with biotin-alkyne by click chemistry and immunoblotted with an anti-biotin antibody. Top panel: non-stimulated cells with cont-si; middle panel: ascorbate-stimulated cells with cont-si; bottom panel: ascorbate-stimulated cells with p180-si.