1. Rab27b Localizes to the Tubulovesicle Membranes of Gastric Parietal Cells and Regulates Acid Secretion 
Jo Suda, Lixin Zhu, Curtis T. Okamoto, Serhan Karvar 
Gastroenterology 
Volume 140, Issue 3, Pages e2 (March 2011)
DOI: /j.gastro
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2. Figure 1
Identification of tubulovesicle proteins. (A) A total of 50 μg of isolated H,K-ATPase–rich tubulovesicles was run. The gel was stained with Coomassie blue. (B) The majority of the bands were identified by tandem MS, representing more than 20 unique proteins.
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3. Figure 2
Identification of Rab27b by MS. In-gel digestion and MS analysis of a ∼25-kilodalton protein band from tubulovesicles were performed as described. (A) Rab27b peptides detected by liquid chromatography tandem MS. Eight peptides, with sequencing information from collision-induced dissociation spectra, appear in bold type. (B) MS spectrum showing isotopic peaks for peptide TTFLYR. A difference of 0.5 daltons between adjacent isotopic peaks indicated that the peptide is doubly charged, and therefore the neutral mass is daltons, matching TTFLYR. (C) Collision-induced dissociation spectrum of peptide TTFLYR. A full series of the C-terminus y-ions from collision-induced dissociation, together with the mass of the intact peptide, specified the amino acid sequence.
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4. Figure 3
Distribution of Rab27b in parietal cells. Density gradient centrifugation was used to isolate a resting and stimulated plasma membrane-rich fraction (PM) from the low-speed P1 pellet and an H,K-ATPase–rich tubulovesicular fraction (TV) from the P3 microsomal pellet. (A and B) All fractions (10μg per lane) were probed for H,K-ATPase and Rab27b. (C) Identification of H,K-ATPase, Rab27b, Rab11, and syntaxin 1 in immunoisolated vesicles. The immunoisolated tubulovesicles (10 μg) were separately probed with both anti–H,K-ATPase β subunit, anti-Rab11, anti-Rab27b, and anti–syntaxin 1.
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5. Figure 4
Localization of Rab27b and H,K-ATPase in parietal cells. Cultured parietal cells were treated with 100 μmol/L cimetidine (Resting) or with secretagogues, 100 μmol/L histamine plus 30 μmol/L IBMX, in the absence (Stimulated) or presence of 5 μmol/L SCH28080, a proton pump inhibitor (Stimulated + SCH). After 30 minutes, the cells were fixed and doubly stained for Rab27b and H,K-ATPase. Bar = 10 μm.
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6. Figure 5
Distribution dynamics of Rab27b in live parietal cells. Primary parietal cells were infected with rAD containing GFP construct only (no Rab27b; upper row) or with rAD expressing YFP-Rab27b WT and constitutively active mutant Q78L and dominant negative mutant N133I adenovirus. Cells were examined in the presence of cimetidine (Resting), stimulated with histamine plus IBMX, either in the absence or presence of the proton pump inhibitor (Stimulated + SCH). Bar = 10 μm.
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7. Figure 6
Intracellular localization of H,K-ATPase in YFP-Rab27b. Primary parietal cells were infected with (A) rAD YFP-Rab27b WT, (B) constitutively active Q78L, and (C) dominant negative N133I adenovirus and maintained in culture as described. The cells were fixed and double immunostained using a fluorescein isothiocyanate–labeled secondary antibody against GFP (which also recognizes YFP) and an antibody against H,K-ATPase detected by rhodamine-labeled secondary antibody. Bar = 10 μm.
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8. Figure 7
YFP-Rab27b N133I inhibits acid secretion. (A) Cells were infected with WT YFP-Rab27b (Rab27b WT), constitutively active YFP-Rab27b Q78L (Rab27b Q78L), and dominant negative YFP-Rab27b N133I (Rab27b N133I) mutants or rAD containing GFP construct only (GFP). Additional cultures of noninfected cells served as untreated control (CONT). Cells were then incubated with cimetidine (REST) or stimulated (STIM), and the [14C]AP accumulation ratio was measured. The data shown here for 5 experiments have been normalized by setting the value for the stimulated control to 100% for each experiment. Values are the mean ± SE of 5 independent experiments, each with duplicate determinations. (B) Measurement of apical membrane vacuoles. Morphometric data were obtained from 3 separate culture preparations in which 50 cells from each category were examined. The cells were infected with control virus, Rab27b WT, and mutants. The apical membrane vacuole diameter was measured in micrometers for all vacuoles that were visible in cross section in resting and stimulated cells. Data are expressed as mean ± SE. **P < .01; the diameter of stimulated cells infected with YFP-Rab27b N133I was compared with that of noninfected, control virus, Rab27b WT, and Rab27b Q78L stimulated cells.
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9. Supplementary Figure 1
Rab27b antibody does not recognize Rab11. Immunoprecipitation of Rab11 using anti-Rab11. Lysates from purified tubulovesicle fraction were separately immunoprecipitated with anti-Rab11. Anti-GFP antibody was used as an immunoprecipitation control. The loading proteins and precipitated proteins eluted from agarose beads were assessed using Western blot analysis with anti–H,K-ATPase β subunit, anti-Rab11, and anti-Rab27b.
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10. Supplementary Figure 2
Intracellular distribution of Rab11 in YFP-Rab27b WT infected parietal cells. Cultured parietal cells were treated with 100 μmol/L cimetidine (Resting) or with secretagogues, 100 μmol/L histamine plus 30 μmol/L IBMX, in the absence (Stimulated) or in the presence of 5 μmol/L SCH-28080, a proton pump inhibitor (Stimulated + SCH). The cells were infected with recombinant YFP-Rab27b WT adenovirus. After 30 minutes, the cells were fixed, immunostained for Rab11 and GFP, and examined by immunofluorescence microscopy.
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11. Supplementary Figure 3
Overexpression of Rab27b in gastric parietal cells. Parietal cells were infected by adenovirus expressing YFP-Rab27b WT, YFP-Rab27b Q78L, and YFP-Rab27b N133I. After 36 hours, cells were lysed, and the samples were separated by SDS-PAGE and analyzed by immunoblot analysis with anti-Rab27b (A). The signals were quantified using ImageJ software. Data shown here are representative of 3 parietal cell culture preparations (B).
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