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Blood, Vol. 109, Issue 12, 5308-5317, June 15, 2007
Roles of RabGEF1/Rabex-5 domains in regulating Fc RI surface expression and FcRI-dependent responses in mast cells
Blood Kalesnikoff et al.
109: 5308
Supplemental materials for: Kalesnikoff et al, Vol 109, Issue 12, 5308-5317
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 46.4 KB)
- Figure S1. IgE+Ag-induced tyrosine phosphorylation events are prolonged in the absence of RabGEF1 (JPG, 47.5 KB)
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(A) RabGEF1+/+ and RabGEF1−/− bone marrow-derived cultured mast cells (BMCMCs) were sensitized for 16 hours with 2 µg/mL IgE, then stimulated with 20 ng/mL DNP for the indicated times. Total cell lysates were subjected to Western blot analysis using  -phosphotyrosine (PY) Abs (4G10). Blots were reprobed with -p44/42 MAPK Abs to show loading. (B) RabGEF1+/+ and RabGEF1−/− BMCMCs sensitized and stimulated as in (A) were subjected to immunoprecipitation with - PY (4G10) Abs and Western blot analysis with -Fc RI  chain or -FcR  chain Abs. An equal volume of each lysate used for immunoprecipitation was subjected to Western blot analysis using -GAPDH Abs to show loading. Results in (A) & (B) are representative of those obtained in 3 or more separate experiments, using 3 or more different populations of RabGEF1+/+ and −/− BMCMCs.
- Figure S2. RabGEF1−/− BMCMC cytosol is deficient in early endosome fusion activity, and expression of WT RabGEF1 in −/− BMCMCs normalizes endosome fusion activity to +/+ levels (JPG, 40.7 KB)
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(A) Fusion activity of indicated concentrations of +/+ (♦) or −/− (■) BMCMC cytosol. Data represent the mean + SEM (n=3) of pooled data from 3 separate experiments performed in duplicate. ***, P<.001 vs. −/− BMCMC cytosol. (B) Fusion activity of indicated concentrations of −/− (■) or WT-expressing −/− (▲) BMCMC cytosol. Data represent mean + SEM (n=2) of pooled data from 2 separate experiments performed in duplicate. **, P < .01; ***, P < .001 vs. −/− BMCMC cytosol. A spontaneously-derived −/− BMCMC line was used for the endosome fusion assays in (B).
- Figure S3. Localization in BMCMCs of WT RabGEF1 (green), Alexa 594-Dextran labeled vesicular structures (red) and Alexa 647 labeled IgE (JPG, 47.5 KB)
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(A, B) Representative merged images of RabGEF1 −/− BMCMCs transiently-transfected (24 hours) with the WT RabGEF1 GFP construct (green), then exposed to (A) Alexa 594- Dextran (red), an endocytic compartment marker, for the final 6 hours, or (B) Alexa 647- IgE (blue) for the final 16 hours plus Alexa 594-Dextran for the final 6 hours. BMCMCs were then induced to adhere to fibronectin coated coverslips, fixed, and imaged by confocal microscopy. In (A), much of the RabGEF1 signal is seen surrounding Dextran-labeled structures (arrowheads). Some Dextran labeled structures appear not to colocalize with RabGEF1 (arrows). In (B), IgE is localized to the cell surface of both transfected and nontransfected cells (arrowheads) and, in a WT RabGEF1 transfected mast cell, to Dextran labeled structures that are surrounded by RabGEF1 (arrows). Bars =|10 µm.
- Figure S4. Localization of RabGEF1 mutants in fibroblasts (JPG, 94.3 KB)
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RabGEF1−/− dermal fibroblasts transiently transfected (24 hours) with the indicated GFP constructs (green) were fixed and examined for EEA1 (red) and Rab5 (blue) colocalization by confocal microscopy using -EEA1 and -Rab5 Abs. White arrowheads indicate representative points of colocalization and higher magnifications of these areas (2.5×) are shown in insets. Bars =|10 µm. Insets show, from left to right, images of the same fields depicting the GFP (RabGEF1) channel, the Alexa 546 (EEA1) channel and Alexa 633 (Rab5) channel.
- Figure S5. Effects of RabGEF1 domains on endosome size (JPG, 106 KB)
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(A) Representative confocal images of −/− fibroblasts used to measure endosome size; 24-36 hours after transfection with the indicated constructs (green), EEA1-positive endosomes (red) were measured. Bars =|25 µm. (B) Average endosome size (>|10 measurements per cell) from two separate experiments (n|=|10-12 cells total) were pooled and bar graphs represent average population endosome size| ± |SEM. +, P<.05; +++, P<.001 vs. WT; ***, P<.001 vs. untransfected.
- Figure S6. Expression of WT RabGEF1, but not D313A, normalizes c-Kit internalization to the levels seen in +/+ BMCMCs (JPG, 56.6 KB)
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RabGEF1+/+ and −/− BMCMCs infected with the indicated lentiviral vectors were starved for 16 hours in Dulbecco modified Eagle medium (DMEM) plus 10% fetal calf serum (FCS), then stimulated with 100 ng/mL SCF for 0 or 3 hours, and surface c-Kit expression was analyzed by flow cytometry. The bar graphs represent the mean + SEM of the percent c- Kit internalization determinations from 3 separate batches of BMCMCs. Percent c-Kit internalization was calculated by subtracting the mean fluorescence intensity at 3 hours from the mean fluorescence intensity at 0 hours and dividing this number by the mean fluorescence intensity at 0 hours (× 100%). +, P<0.05; ++, P<0.01 vs. +/+ “empty” values; **, P<0.01 vs. −/− “empty” values.
- Figure S7. The ZnF domain of RabGEF1 mediates ubiquitination in vitro (JPG, 80 KB)
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(A) In vitro ubiquitination assays with WT RabGEF1 GST fusion proteins were performed in the presence of Ub and the indicated Ub-conjugating enzyme (E2), and the presence or absence of Ub activating enzyme (E1). Samples were analyzed by Western blot using -Ub Abs. (B) In vitro ubiquitination assays with WT or mutant RabGEF1 GST fusion proteins were performed in the presence of Ub, UbcH5a (E2), and Ub activating enzyme (E1). Samples were analyzed by Western blot using -Ub, -RabGEF1, and -RabGEF1 (Ac-KSER) Abs. The -RabGEF1 (Ac-KSER) polyclonal Ab generated by our laboratory (Tam et al, 2004) was used to examine RabGEF1 immunoreactivity of the ubiquitinated CT2 fusion protein because this fusion protein lacks the consensus sequence for the -RabGEF1 Ab generated by BD (shown in Fig. 2A). Conversely, the NT fusion protein lacks the consensus sequence for the -RabGEF1 (Ac-KSER) Ab (Fig. 2A). * = monoubiquitinated RabGEF1.
- Figure S8. Rabaptin-5 mRNA levels are similar in RabGEF1+/+ and −/− BMCMCs as assessed by semi-quantitative RT-PCR (JPG, 41 KB)
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Equal quantities of cDNA from RabGEF1+/+ and −/− BMCMCs were PCR amplified using Rabaptin-5en (top panels) or GAPDH- (bottom panels) specific primers for the indicated number of cycles. PCR products were separated on a TAE agarose gel and stained with ethidium bromide, as described in Materials and Methods.
- Figure S9. Binding of WT or mutant RabGEF1 to activated Ras (JPG, 62.6 KB)
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RabGEF1−/− BMCMCs infected with the indicated lentiviral vectors were sensitized for 16 hours in DMEM + 10% FCS containing 2 µg/mL IgE, then stimulated for 15 minutes with 20 ng/mL DNP. Lysates were subjected to pull-down assays with a GST fusion protein containing the Ras-binding domain (RBD) of Raf-1 (right panels), which binds to GTP-loaded or active Ras, using the EZ Detect Ras Activation Kit (Pierce) as described previously (Tam et al., 2004). TCLs (left panels) and pull-downs were analyzed by Western blot using -HA, -RabGEF1 and -Ras Abs.
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