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Blood, Vol. 105, Issue 10, 4088-4095, May 15, 2005
Protein-4.2 association with band 3 (AE1, SLCA4) in Xenopus oocytes: effects of three natural protein-4.2 mutations associated with hemolytic anemia
Blood Toye et al.
105: 4088
Supplemental materials for: Toye et al, Volume 105, Issue 10, 4088-4096
Files in this Data Supplement:
- Figure S1. Investigation of the effects of protein 4.2 on the levels of band 3 at the oocyte surface using a protease accessibility assay (JPG, 230 KB)
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To investigate the effects of protein 4.2 on the levels of band 3 at the oocyte surface, oocytes were either labeled with 35[S]-methionine (A) or left unlabeled (B) and allowed to express protein for 48 hours and then treated with chymotrypsin for 1 hour at 4°C, as described previously by Groves and Tanner30 and Kanki et al (Kanki T, Young MT, Sakaguchi M, Hamasaki N, Tanner MJ. The N-terminal region of the transmembrane domain of human erythrocyte band 3: residues critical for membrane insertion and transport activity. J Biol Chem. 2003;278:5564-5573). (A) 0.5 ng band-3 cRNA alone or 0.5 ng band-3 cRNA and 1.5 ng normal protein 4.2 or protein-4.2 variant cRNA (4.2 Nippon is indicated by 4.2N; 4.2 Tozeur, by 4.2T; and 4.2 Komatsu, by 4.2K) were injected into oocytes. B3 was immunoprecipitated from the homogenate of the equivalent of 5 oocytes using Bric 155, and immunoprecipitations were carried out in duplicate. The immunoprecipitates were separated on 8% SDS PAGE gel. The position of the whole B3 and 35-kDa cleaved band are indicated. The levels of B3 at the oocyte surface appear to decrease on coexpression of either normal protein-4.2 cRNA or protein-4.2 Nippon cRNA, even though coexpression of these proteins enhanced band-3 anion transport in the oocytes. In contrast, protein 4.2 Tozeur and protein 4.2 Komatsu had no effect on the cell surface surface expression of band 3. We were unsuccessful in obtaining reproducible results using 0.5 ng band-3 cRNA, despite several attempts, due to the levels of chymotrypsin-cleaved immunoprecipiated protein being too variable at these low band-3 expression levels. This limitation of the proteolysis assay below 1.5 ng B3 cRNA has been previously reported.35 (B) 5 ng band-3 cRNA alone or 5 ng band-3 cRNA and 1.5ng GPA cRNA or 5 ng band-3 cRNA and normal protein 4.2 or protein-4.2 variant cRNA (4.2 Nippon is indicated by 4.2N; 4.2 Tozeur, by 4.2T; and 4.2 Komatsu, by 4.2K) were injected into oocytes. Groups of 10 oocytes were homogenized and centrifuged twice at 13|000 rpm for 5 minutes to remove cell debris. The homogenate was then separated on a 12% SDS-PAGE gel; band 3 and band fragments were visualized by Western blotting using the anti—C-terminal band-3 monoclonal antibody Bric 155. Each lane represents the protein from 5 oocytes. The proportion of band 3 cleaved at the oocyte surface by chymotrypsin to generate the 35-kDa fragment is indicated under each lane. These figures were determined using scanning densitometry and were calculated from a least 3 separate experiments. Most lanes in panel B show a third band running at 55 kDa, which is assumed to be the membrane domain of band 3 (generated by internal cleavage of band 3 between the membrane and cytoplasmic domain during band-3 expression in oocytes; Kanki et al, 2003). This experiment shows that the coexpression of protein 4.2 with B3 at high band-3 cDNA concentrations (5 ng) does not alter the levels of B3 expressed at the cell surface. This is consistent with the lack of significant effect of protein 4.2 on the anion at high band-3 concentrations (5 ng) under these conditions. As previously reported, the coexpression of GPA cDNA with band-3 cDNA clearly enhances band-3 levels at the cell surface (Groves and Tanner30; Kanki et al., 2003).
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