Fig 1: Human RAP1, GFAPɛ and PS1 interact with one another in vitro. a) Proteins were expressed in BL21 and BL21(DE3) cells. MBP-PS11–85 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.
Fig 2: RAP1 and GFAPɛ colocalize with subunits of the γ-secretase complex. SH-SY5Y cells were grown, fixed, and probed with antibodies as indicated in each panel. Representative images are shown that include images of each fluorophore individually with a larger, merged image. The box in the merged images indicates the region that is increased in size for the inset at the top right corner. Arrows in the inset indicate examples of colocalization. a) RAP1 and GFAPɛ colocalize. Cells were probed with DAPI, antibodies to RAP1 and antibodies to GFAPɛ. b) RAP1 colocalizes with the γ-secretase subunit PSENEN. Cells were probed with DAPI, antibodies to RAP1, and antibodies to PSENEN. c) RAP1 and GFAPɛ colocalize with the γ-secretase subunit PS1. DAPI stain was not used in this image. Cells were probed with antibodies to PS1, antibodies to RAP1 and antibodies to GFAPɛ.
Fig 3: GFAPɛ coimmunoprecipitates with RAP1 from human SH-SY5Y neuroblastoma cells. SH-SY5Y cells were grown and lysed. Rabbit antibodies to RAP1 or non-specific rabbit antibodies were used to precipitate proteins that were eluted and resolved by SDS-PAGE. The panel on the left shows a membrane probed with the antibodies to RAP1. The panel on the right shows an identically loaded membrane probed with antibodies to GFAPɛ.
Fig 4: RAP1 and GFAPɛ affect the activity of human γ-secretase expressed in yeast. a) Strain MY14 (expressing C99-Gal4p) was transformed with pBEVY-T and pBEVY-L vectors or the pBEVY plasmids expressing the four subunits of the γ-secretase complex. Four independent transformants of each were grown for use in an α-galactosidase activity assay. The bars on the graph represent the average α-galactosidase activity with error bars representing the standard error of the mean. The P-value was determined using a one-tailed student's t-test (***P < 0.001). b) Strain MY14 (expressing C99-Gal4p) with pBEVY-T and pBEVY-L vectors or the pBEVY plasmids expressing the four subunits of the γ-secretase complex were transformed with the pBEVY-U vector, or pBEVY-U expressing RAP1, GFAPɛ, or both RAP1 and GFAPɛ. Three independent transformants of each were assayed. Cells were grown in SC-trp-leu-ura medium. Cells were spotted directly onto SC-trp-leu-ura medium with X-α-gal (panels on right) or diluted before plating the 1:10 and 1:100 dilutions on SC-trp-leu-ura (control) medium (left panels) or SC-trp-leu-ura-ade-his medium (center panels). The dilutions are indicated above the images, and the sample numbers are indicated at the bottom. c) Strain MY14 (expressing C99-Gal4p) with the pBEVY plasmids expressing the four subunits of γ-secretase were transformed with the pBEVY-U vector, or pBEVY-U expressing RAP1, GFAPɛ, or both RAP1 and GFAPɛ. Three independent transformants of each were used, and three technical replicates for each assayed for α-galactosidase activity. The technical replicate values were averaged to represent each of the biological replicates. The bars on the graph represent the average α-galactosidase activity of the biological replicates relative to the average activity of the control for simplicity. Error bars represent the standard error of the mean of the biological replicates. The P-values were determined using a nested, one-way ANOVA with a Tukey's multiple comparisons test (**P < 0.01; ***P < 0.001; ns, not significant). d) U251 cells were transfected with pLPC vector or pLPC-hRAP1 FL that overexpresses RAP1. Six wells transfected with each plasmid were used to measure Aβ40 and Aβ42. Two technical replicates for each were measured using ELISA. The bar graphs represent the average concentration of the peptides in the media. Error bars represent the standard error of the mean of the biological replicates. P-values were determined using a two-tailed student's t-test (**P < 0.01; ***P < 0.001).
Fig 5: Human RAP1 interacts specifically with the epsilon isoform of glial fibrillary acidic protein (GFAPɛ) in the Y2H system. a) The domain organization of the GFAPɛ protein is shown at the top of the image. GFAPɛ consists of α-helical rod-domains (grey boxes) flanked by a head and tail domain. The thick portion of the tail domain is the segment unique to the GFAPɛ isoform. Below the diagram are lines depicting the segments of GFAPɛ that were isolated as Gal4p activation domain fusions from the Y2H library. b) GFAPα and GFAPɛ are compared. The boxes at the top depict the exons included for the differentially spliced forms, which encode distinct tail regions. Below is a comparison of the protein sequence differences between the isoforms. The isoforms are identical in the N-terminal 390 amino acids but differ in their C-termini (42 amino acids for GFAPα and 41 amino acids for GFAPɛ). c) Y2H Gold cells transformed with plasmids show an interaction between RAP1 and GFAPɛ but not with GFAPα. Three biological replicates (independent transformants) of each were grown in liquid SC-trp-leu medium overnight before being diluted 1:10 and spotted onto SC-trp-leu (control) medium and SC-trp-leu-ade-his medium. GAL4DBD fusions are shown in the first column, and GAL4AD fusions are shown in the second column. The GFAP fusions used are based on the smallest fragment isolated in the Y2H screen, GFAPα 204–432 and GFAPɛ 204–431. d) Y2H Gold cells were transformed with RAP1 and the full-length GFAPɛ protein. The experiment was set up as in (c), except GFAPɛ 204–431 and GFAPɛ 1–431 were tested for interaction with RAP1.
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