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Blood, Vol. 111, Issue 11, 5380-5389, June 1, 2008
Band 3 Courcouronnes (Ser667Phe): a trafficking mutant differentially rescued by wild-type band 3 and glycophorin A
Blood Toye et al.
111: 5380
Supplemental materials for: Toye et al
Files in this Data Supplement:
- Figure S1. BRIC6 binding to control and S667F-B3 cells (JPG, 49 KB)
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Panels A-C show MDCK1 cells and Panels D-M stably expressing S667F-kB3 that were grown on cover slips. The cells in Panels A-C and H-M were washed, incubated with the extracellular anti-band 3 antibody BRIC6 for 1 hour and then fixed and stained with rbB3ct antibody as outlined in the methods section. Panels D-G were double stained with BRIC170 and rbB3ct antibodies to compare the abilities of these antibodies to detect a range of S667F-kB3 protein expression levels. Panel A-C is a control panel showing that no FITC-BRIC6 binds MDCK1 cells after 1 hour incubation with FITC-BRIC6 (panel B) and rbB3ct has a slight background reaction with the nucleus (panel C) but this background is not present when using cells expressing kB3 protein. Panel D and E show the difference between BRIC170 and rbB3ct in detecting low levels of S667F-kB3. These differences are not evident at higher levels of cellular expression (Panels F and G). Panels H, J and L show a range of S667F-B3 detected by rbB3Ct and shows the relationship between levels of FITC-BRIC6 detected on cells (panels I, K, M) is correlated with higher expression of S667F-kB3 in MDCK cells. Scale bar: 30 µM.
- Figure S2. DNA analysis (JPG, 41 KB)
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The DNA sequence between bases 1984-2015 of SLC4A1 are shown. Base 2000 is boxed and is “C” in control DNA, “T” in the proband’s DNA. Both bases (C and T) are present in the heterozygous parents. M: mother; P: proband; F: father; C: control.
- Figure S3. Radiolabeling of kB3 and S667F-kB3 in MDCK1 cells (JPG, 44.6 KB)
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Non-polarized cells were preincubated with sodium butyrate to increase protein expression and labeled with 200 µCi/ml Easy-Tag™ 35S-methionine for 30 minutes and pulse chased for the times indicated as previously described (ref 17). Immunoprecipitates were then prepared using anti-B3 BRIC155 bound to protein A beads, washed in IP buffer and then run on 8% SDS PAGE gels. The upper panel shows kB3 and the lower panel S667F-kB3 protein expression over the time course of the pulse chase. Because kB3 is expressed at a higher level in MDCK1 cells we loaded half the material for the IP of each time point for comparison with S667F-KB3. The results are representative of three independent experiments. S667F-kB3 is unstable when expressed in MDCK1 cells, with the majority of the mutant protein being turned over within 4-6 hours and the protein being totally absent by 12 hours.
- Figure S4. Co-expression of wild-type B3/kB3 with various band 3 mutants (JPG, 75.2 KB)
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Various mutant cRNAs were expressed alone or co-expressed with wild-type B3/kB3 at the concentrations indicated in Xenopus oocytes. DNDS-sensitive chloride uptake (1 hour) was measured 24 hours after injection with the cRNA using groups of 12-15 oocytes. Results are shown as means ± SEM. Panel A shows the effects of co-expression of wild-type kB3 with G701DkB3, DelV850kB3 or A858DkB3 dRTA mutants previously reported in Bruce et al, B.J. 2000 to give an indication of transport gained when wild-type protein is combined with other known B3 mutants. The predicted amount of activity for each wild-type B3/mutant combination is shown, and represents the expected contribution of 50% kB3 and 50% mutant chloride influx assuming that each population is independent. This shows that all three mutants give approximately the predicted transport when co-expressed with wild-type kB3.
We have also included the influx gained from expressing 0.75 ng kB3 alone but it should be noted that this represents a situation where the wild-type has no competition for the oocyte translational machinery (compared with co-injection with a mutant cRNA) and so gives influx higher than 50% of 1.5 ng wild-type kB3. (Representative of two independent experiments). Panel B shows the effects of co-expression of wild-type B3 with S667F-B3 or G701D-B3 with the predicted influx indicated. As reported in this manuscript (Figure S4E) S667F-B3 is substantially rescued by co-expression of wild-type protein. However, wild-type protein did not appear to significantly rescue G701D. (Representative of two independent experiments).
- Figure S5. Cell surface biotinylation studies on oocytes co-expressing wild-type kB3 and S667F-B3 (JPG, 41.5 KB)
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A total of 5 ng B3, kB3 or S667F-B3 alone or 5 ng each of wild-type and S667F-B3 was injected into Xenopus oocytes incubated for 24 hours. 5 ng of each was chosen to ensure that enough protein was present for detection on Western blots. The truncated wild-type kB3 protein was used to enable us to distinguish the wild-type protein band from the mutant protein on SDS PAGE gels. A biotinylation assay was then conducted as outlined in materials and methods section. The biotinylation assay used material from 3 immunoprecipitations (10 oocytes per IP) and the biotinylated fraction from this pooled material isolated using strepavidin beads (representative of 2 separate experiments). 1/20th of the input from the 3 IPs is also shown. When expressed alone there is low levels of S667F-B3 at the cell surface as reported in Figure S4D. The data shows similar levels of expression of S667F-B3 and wild-type kB3 at the plasma membrane are achieved when both proteins are co-expressed.
- Figure S6. Higher resolution and magnification images of S667F-kB3 expression in non-polarized MDCK1 cells (JPG, 127 KB)
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Panels A-L show MDCK1 cells stably expressing normal kB3 or mutant S667F-kB3 grown on cover slips and fixed. Panels A-L show a comparison of kB3 and S667F-kB3 localization detected using Bric170, with intracellular markers for the ER (Calnexin) or TGN (TGN 38). kB3 had only a partial overlap with the ER marker (merge panel C), some over lap with TGN38 (merge panel I) and the majority of the protein is at plasma membrane as previously reported.17 The majority of mutant S667F-kB3 immunoreactive protein overlapped with the calnexin (merge panel F) but there was some overlap with the TGN38 staining (merge panel L), suggesting that a small proportion of the protein reaches the late stages of the secretory pathway. Scale bar: 15 µm.
- Figure S7. Polarized expression of S667F-kB3 in MDCK1 cells (JPG, 128 KB)
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Panels show MDCK1 cells stably expressing kB3 or S667F-kB3 that were allowed to polarize for 3 days, their protein expression induced with sodium butyrate and fixed as described in the materials and methods. The cells were then double labeled with anti-band 3 mouse monoclonal BRIC170 and a rabbit antibody to beta catenin, the bound antibodies detected with suitable goat anti-mouse or rabbit secondary antibodies and imaged using confocal microscopy. The upper panel (XY) is a view parallel to the epithelium showing the BRIC170, beta catenin and merged image. The images below (XZ) show a perpendicular view of BRIC170, beta catenin and the merged image of the same epithelium, as represented by the white line in the XY image. kB3 co-localized with the basolateral marker beta catanin, but S667F-kB3 immunoreactive protein did not localize to the basolateral membrane in polarized cells. Scale bar: 30 µm.
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