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Blood, Vol. 112, Issue 10, 4148-4157, November 15, 2008
Rabaptin-5 regulates receptor expression and functional activation in mast cells
Blood Rios et al.
112: 4148
Supplemental materials for: Rios et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 833 KB)
- Figure S1. Protein and mRNA levels in Rabaptin-5–deficient cells (JPG, 66.1 KB)
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(A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were lysed in sample buffer, resolved by SDS-PAGE and prepared for Western blot analysis as described in the Materials and Methods. Membranes were immunoblotted with the indicated antibodies. Results from three pairs of shC- or shR-treated BMCMCs are depicted. GADPH levels are shown as loading controls. (B) RNA isolated from shC- or shR-treated BMCMCs was reverse transcribed as described in the Materials and Methods. Bar graph represents relative FcεRI αmRNA levels normalized to GAPDH expression from three pairs of shC- or shR-treated BMCMCs evaluated by quantitative PCR. No statistically significant difference (i.e., p<0.05) was noted when values for shR-treated cells were compared to those for shC-treated cells (assigned a hypothetical value of one) by a one sample t-test.
- Figure S2. Alexa 633 BSA uptake and autofluorescence in control and Rabaptin-5–deficient BMCMCs (JPG, 37.9 KB)
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(A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were pulsed with Alexa 633-labeled BSA for 20 min, washed and chased with unlabeled BSA for the indicated times. Cell-associated Alexa 633 mean fluorescence intensities (MFI) were measured by flow cytometry at the indicated time after the pulse. Dead cells were excluded from analysis by propidium iodide gating. Bar graph represents data pooled from four separate shC and shR BMCMC pairs. No statistically significant differences were noted when means from shC-and shR-treated BMCMCs were compared at each time point, but a statistically significant decrease in MFI over time (compared to values for time0) was noted in each group (*, p <0.05 vs MFI at time 0, by Student’s t-test, 2-paired). (B) shC- or shR-treated BMCMCs were removed from culture and washed, and autofluorescence was analyzed by flow cytometry. FL-1, FL-2 and FL-4 indicate the filter channels used to assess fluorescence emission from 515–545 nm, 564–606 nm and 653–669 nm, respectively. For autofluorescence in the FL-1 channel (GFP channel), cells were infected with a pLL3.7 construct that expresses the Cyan fluorescent protein, not GFP, to asses intrinsic autofluorescence in this channel. Notably, in cells infected with the standard GFP-expressing constructs, GFP expression was similar in shC-and shR-treated BMCMCs. Data represent mean fluorescence intensities (MFIs) from three or more pairs of shC- or shR-treated BMCMCs. No statistically significant differences (i.e., p <0.05)were noted between values for shC-vs. shR-treated cells when assessed by Student’s t-test, 2-tailed.
- Figure S3. Intracellular FcεRI localization is perturbed after brefeldin A treatment or in cells lacking the common FcR γchain (JPG, 65.8 KB)
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(A) The relative subcellular localization of each of the three chains that constitute functional FcεRI was assessed by immunofluorescence staining of the FcεRIα chain, FcεRIβ chain, and FcεRIγ chain. Cells were prepared for confocal microscopy as described in the Materials and Methods. (B and C) Subcellular distribution of FcεRIα (green) and the endosplasmic reticulum marker calnexin (red) after a 12 h exposure of BMCMCS to 100 µg/ml brefeldinA (B) or in cells that lacked the common gamma chain for the Fc receptors (FcRγ−/−) (C). Arrowheads in (B) and (C) indicate points of FcεRIα and calnexin protein co-localization. Scale bars indicate 7.5 µm.
- Figure S4. Efficient release of FcεRI from intracellular stores, and surface maintenance, requires Rab11 activity (JPG, 74.9 KB)
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(A) BMCMCs were transiently transfected for 24 h with Rab11-GFP or the dominant negative form of Rab11 (Rab11 S25N-GFP), stained with the indicated antibodies and imaged by confocal microscopy. Results are representative of three separate experiments. Insetsdepict magnification of regions in the given field. Scale bars indicate 7.5 µm. (B) BMCMCs were transiently transfected with wild type Rab11 (Rab11 WT GFP-black filled bars in bar graph), dominant negative Rab11 (Rab11 S25N GFP-open bars in bar graph), or dominant negative Rab4 (Rab4 S22N GFP-dashed bars in bar graph) (a gift from Dr. Marci Scidmore, Cornell), for 24 h after which cells were fixed and analyzed by flow cytometry for surface or total FcεRIα. Dot plots are representative of four experiments from BMCMCs cultured from four separate mice. The mean fluorescence intensities (MFI) from low, medium and high GFP-expressing cells were normalized to reflect percent MFI relative to MFI of GFP-negative cells. Data pooled from the four experiments are presented in the bar graph. *, p <0.05 when compared to a hypothetical value of 100 by a one-sample t-test.
- Figure S5. IgE accumulates in Rab9 positive structures after sensitization and does not undergo recycling at rest (JPG, 74.5 KB)
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(A) BMCMCs were sensitized with 2 µg/ml of IgE for 18 h, fixed, stained for surface or total IgE and associated fluorescence was measured by flow cytometry. MFIs were divided by the MFI of surface staining to generate % of surface staining. Data pooled from three separate experiments are presented in the bar graph. (B) Mast cells sensitized as in (A) were induced to adhere to FN and subsequently stained for IgE (top panel) or were sensitized with Alexa 647-labeled IgE (IgE-647) for 18 h, induced to adhere to FN (bottom panel) and assessed for associated fluorescence by confocal microscopy. (C) BMCMCs were transiently transfected for 18 h with Rab9-GFP, and sensitized with IgE-647 for 6 or 16 h, prepared and imaged by confocal microscopy as described in (B). Insets depict magnified Rab9-GFP (upper) and IgE-647 (lower) channels of the regions marked by the dashed boxes. (D) Mast cells were sensitized for 18 h with 1 µg/ml of IgE labeled with a cleavable form of biotin. Surface associated biotin was removed and cells were allowed to recover for the indicated times after which the cells were stained with APC-conjugated streptavidin. Data were pooled from three separate experiments and are expressed as associated MFI divided by MFI at time 0. *,p <0.05; ***, p <0.001 when compared to a hypothetical value of 100 by a one sample t-test. Scale bars indicate 7.5 µm.
- Figure S6. Effect of Rabaptin-5 deficiency and intracellular trafficking inhibitors on mast cell β1 and FcεRI surface expression (JPG, 43 KB)
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(A) Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were exposed to 50 µM primaquine (PQ) for the indicated times and then levels of surface FcεRI were assessed by flow cytometry. Data were pooled from three pairs of shC- or shR-treated BMCMCs. (B) β1 integrin surface stability was measured in shC- or shR-treated BMCMCs by exposing cells to 100 µg/ml brefeldin A (BFA) or 1.5 µg/ml cycloheximide (CHX) for the indicated times. Surface β1was then assessed by flow cytometry. Non-viable cells were excluded by propidium iodide staining. Mean fluorescence intensities (MFIs) relative to the MFI at time 0 was calculated to generate the percentages shown for the indicated time points. Data were pooled from three pairs of shC- or shR-treated BMCMCs. *, p <0.05 vs a hypothetical value of 100 for the indicated treatment by a onesample t-test. +, p <0.05 vs. corresponding values for shC-treated cells at the indicated time by Student’s t-test, 2-tailed.
- Figure S7. The Rabaptin-5 interacting proteins GGA1 and γ-adaptin are localized normally in the absence of Rabaptin-5 (JPG, 83.9 KB)
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Control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs were processed for confocal microscopy as described in the Materials and Methods. GGA1 (red)was localized with a rabbit anti-GGA1 antiserum generously provided by Dr. Margaret Robinson (University of Cambridge) and γ-adaptin (green) was localized with a mouse monoclonal anti–γ-adaptin (BD Biosciences). Results are representative of the similar findings we obtained in the three separate experiments we performed. Scale bars indicate 7.5µm.
- Figure S8. Viability of control and Rabaptin-5–deficient BMCMCs (JPG, 32.3 KB)
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Viability of control (shC) or Rabaptin-5 (shR) shRNA-treated BMCMCs that had been cultured in DMEM + 10% FCS + 10 ng/ml IL-3 (0 h) or for 48 or 72 h after withdrawal of IL-3 and supplementation of the culture medium with the indicated growth factors: NS (DMEM + 10% FCS), IL-3 (DMEM + 10% FCS + 1 ng/ml IL-3), SCF (DMEM + 10% FCS + 2.5 ng/ml SCF), IgE (DMEM + 10% FCS + 2.5 µg/ml mouse monoclonal IgE clone SPE-7, Sigma). Non-viable cells were identified by flow cytometry as annexin-V positive (BD Bioscience), GFP low/negative cells. Data were pooled from four pairs of shC- or shR-treated BMCMCs. *, p <0.05, **, p< 0.01 vs. corresponding values for shC-treated BMCMCs by Student’s t-test, 2-tailed.
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