(A) Schematic diagram of tubular ER isolation. P1K, P20K, and P100K represent the pellets of solutions centrifuged at 1000 x g, 20,000 x g, and 100,000 x g, respectively. S100K is the supernatant of the solution centrifuged at 100,000 x g. (B) Microsome preparation by step centrifugation. Samples from each step were immunoblotted using the indicated antibodies. Total, total cell lysates; S1K, S20K, S100K, and P100K are defined as in (A). (C) Sec63p-GFP/Yop1p-Strep or Sec63p-HA/Yop1p-2xFlag was co-transformed into yeast cells. Microsomes were prepared and subsequent immunoprecipitation (IP) performed using anti-Flag agarose beads. The samples were analyzed by SDS/PAGE and immunoblotting (IB). (D) As in (C), but with the addition of other organelle makers. The data shown in B-D are representative of at least three repetitions.