Figure 1.
Rab3b- and Rab27a-APEX2 fusion proteins localize to WPB and biotinylate proteins in close proximity. (A-B) HUVEC were transfected with either Rab3b-FLAG-APEX2 or Rab27a-FLAG-APEX2 constructs. APEX2 fusion proteins were stained by anti-FLAG antibodies (cyan), VWF used as a WPB marker was visualized by anti-VWF antibodies (yellow), and biotinylated proteins were labeled by AF647-streptavidin (magenta). Images on the left side show a merge of the whole cell (A), whereas images on the right show the boxed areas at higher magnification (B). Both Rab3b- and Rab27a-FLAG-APEX2 localize to WPB, and no streptavidin signal is visible in cells before BP/hydrogen peroxide treatment (−BP). After treatment with BP and hydrogen peroxide (+BP), cells show a notably enhanced streptavidin signal that is concentrated in the proximity of WPB. Nuclei are shown in dark blue. Scale bars, 10 µm. (C) Streptavidin pulldowns from lysates of BP/H2O2-treated HUVEC expressing Rab27a-FLAG-APEX2, Rab3b-FLAG-APEX2, or FLAG-APEX2-NES, respectively, were carried out in triplicates, and proteins enriched were identified by tandem MS. Proteins that were present in all 3 replicates (a, b, c) of at least 1 sample group (Rab27a-FLAG-APEX2, Rab3b-FLAG-APEX2, or FLAG-APEX2-NES) were included in the calculation of relative protein enrichment by ANOVA. Because of the very low yield of biotinylated proteins from the nontransfected cells, this sample group was not included. Proteins with P < .02 were considered significantly enriched, leading to the identification of 288 proteins that were clustered into different groups according to their similar enrichment pattern in the different samples. This pattern is visualized by showing high relative enrichment in red and reduced amounts of protein in green. A total of 183 proteins were identified as highly enriched in the WPB-associated samples compared with cytosolic proteins biotinylated by FLAG-APEX2-NES: 19 proteins enriched only in pulldowns from Rab3b-FLAG-APEX2 expressing cells, 6 proteins enriched only in pulldowns from Rab27a-FLAG-APEX2 expressing cells, and a large cluster of 158 proteins enriched in pulldowns from both Rab27a- and Rab3b-FLAG-APEX2 expressing cells (supplemental Tables 1-3).

Rab3b- and Rab27a-APEX2 fusion proteins localize to WPB and biotinylate proteins in close proximity. (A-B) HUVEC were transfected with either Rab3b-FLAG-APEX2 or Rab27a-FLAG-APEX2 constructs. APEX2 fusion proteins were stained by anti-FLAG antibodies (cyan), VWF used as a WPB marker was visualized by anti-VWF antibodies (yellow), and biotinylated proteins were labeled by AF647-streptavidin (magenta). Images on the left side show a merge of the whole cell (A), whereas images on the right show the boxed areas at higher magnification (B). Both Rab3b- and Rab27a-FLAG-APEX2 localize to WPB, and no streptavidin signal is visible in cells before BP/hydrogen peroxide treatment (−BP). After treatment with BP and hydrogen peroxide (+BP), cells show a notably enhanced streptavidin signal that is concentrated in the proximity of WPB. Nuclei are shown in dark blue. Scale bars, 10 µm. (C) Streptavidin pulldowns from lysates of BP/H2O2-treated HUVEC expressing Rab27a-FLAG-APEX2, Rab3b-FLAG-APEX2, or FLAG-APEX2-NES, respectively, were carried out in triplicates, and proteins enriched were identified by tandem MS. Proteins that were present in all 3 replicates (a, b, c) of at least 1 sample group (Rab27a-FLAG-APEX2, Rab3b-FLAG-APEX2, or FLAG-APEX2-NES) were included in the calculation of relative protein enrichment by ANOVA. Because of the very low yield of biotinylated proteins from the nontransfected cells, this sample group was not included. Proteins with P < .02 were considered significantly enriched, leading to the identification of 288 proteins that were clustered into different groups according to their similar enrichment pattern in the different samples. This pattern is visualized by showing high relative enrichment in red and reduced amounts of protein in green. A total of 183 proteins were identified as highly enriched in the WPB-associated samples compared with cytosolic proteins biotinylated by FLAG-APEX2-NES: 19 proteins enriched only in pulldowns from Rab3b-FLAG-APEX2 expressing cells, 6 proteins enriched only in pulldowns from Rab27a-FLAG-APEX2 expressing cells, and a large cluster of 158 proteins enriched in pulldowns from both Rab27a- and Rab3b-FLAG-APEX2 expressing cells (supplemental Tables 1-3).

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