Fig. 2.

Human RAP1, GFAPɛ and PS1 interact with one another in vitro. a) Proteins were expressed in BL21 and BL21(DE3) cells. MBP-PS1_1–85 and the His₆-tagged GFAP proteins (amino acids 204—end) were purified prior to being added to glutathione beads alone or beads prebound with GST-RAP1. The input lanes show the purified proteins (lanes 1, 3, and 4) or GST-RAP1 lysate (lane 2). Eluates from the glutathione beads are in lanes 5 through 13. Proteins included in each sample are listed above the blot image. The blot was probed simultaneously with primary antibodies to each of the tags, and IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image. b) His₆-tagged GFAP proteins (amino acids 204—end) were expressed together with GST-RAP1 (lanes 1 through 4) or individually (lanes 5 through 8). Lysates (inputs) were mixed with glutathione beads, and eluted proteins were resolved on the gel. The blot was simultaneously probed with antibodies to GST and His₆, and IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image. c) The GST and His₆-tagged proteins were expressed together in HMS174(DE3) cells. Where indicated, a third protein expressed separately in HMS174(DE3) cells was added. Only eluates are shown for simplicity. The blot was simultaneously probed with antibodies to GST, His₆, and MBP. IRDye secondary antibodies were used for detection. The protein bands are labeled to the right of the blot image.