Figure 3

Immunoblot analysis of samples fractionated on 10-30% Optiprep density gradients. A - Fractionation of microsomes prepared from heads. BiP, marking the ER, peaks in the center of the gradient in fractions 9-11. ATP synthase shows that residual mitochondrial membrane is slightly heavier than the ER with a peak in fraction 7, but also shows a small presence in higher fractions at the top of the gradient. Nervana is seen as three prominent bands. The lowest (arrowhead) represents minimally processed protein and is restricted to the ER. The mature glycosylated forms (bracket) represent post-Golgi compartments and the plasma membrane. See [28] for a description of Nervana glycosylation patterns. B - Fractionation of microsomes prepared from 0-15 hr embryos. BiP, marking the ER, peaks slightly lower than the ER from head gradients in fractions 7-9. ATP synthase again peaks in fraction 7, and again has a small presence in higher fractions at the top of the gradient. α-Spectrin has a peak in the Golgi region (fractions 3-4), extends through the ER and shows a distinct peak at the top of the gradient. Labeling: 1-20 - fraction number from bottom to top of tube; all other labels are as in figure 2. Loading: An equal volume of all fractions were loaded, except the peak protein fractions (4-6) where 50 μg of protein was loaded to prevent overloading.