Figure 3.

Subcellular localization of Srp102p. Extracts from cells (srp102::HIS3 bearing pSO459) were fractionated as detailed in Materials and Methods. Equivalent amounts (0.5 OD₆₀₀) of each fraction were analyzed by Western blot using anti-HA monoclonal antibodies, affinity-purified anti-Srp101p antibodies, or anti-Sec61p antibodies. Lane 1, total cell lysate(T); lane 2, low-speed supernatant (LS); lane 3, low-speed pellet (LP); lanes 6–13 contain the supernatant (lanes 6, 8, 10, and 12 [S]) or the pellet (lanes 7, 9, 11, and 13 [P]) after a portion of the low-speed supernatant was diluted with lysis buffer (lanes 4 and 5 [HS and HP]) or adjusted to a final concentrations of 600 mM potassium acetate (lanes 6 and 7 [KOAc]), 1.6 M urea (lanes 8 and 9 [Urea]), 0.5% Triton X-100 (lanes 10 and 11 [TX-100]), or 0.1 M Na₂CO₃ (lanes 12 and 13, [pH 11.5]) and centrifuged at 100,000 g. Only the relevant portion of each blot is shown. For comparison, the same fractions were also blotted with anti-Srp101p, a peripheral membrane protein, and anti-Sec61p, a previously characterized integral membrane protein of the ER.