JCS03101 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Relative abundance of mRNA encoding R664X mutant AE1 in bone marrow cells from mutant animals. A, total amounts of mRNA for AE1 (closed squares) and protein 4.1 (open squares) were determined for normal (N, n=3), heterozygous (He, n=2), and homozygous (Ho, n=2) animals by quantitative PCR and described as copy number per 1,000 copies of GAPDH mRNA. Data are expressed as means ± s.d. for N and as mean values for He and Ho. The number at the right indicates the relative value of AE1 mRNA/protein 4.1 mRNA. B and C, relative abundance of mRNA encoding mutant protein. PCR amplification of genomic DNA (gDNA) or bone marrow cell cDNA (cDNA) showed exponential increases of ³²P-labeled products of the 107-bp fragment from the 22nd through 28th cycles. A typical autoradiogram of ³²P-labeled PCR fragments obtained after 26 cycles of PCR amplification followed by digestion with Dra III is shown (B). When gDNAs from heterozygous animals were amplified, in which the mutant allele comprised 50% of the template, densitometric intensities of the 62-bp fragment generated by digestion of the products from the mutant allele with Dra III were 23-25% of the total. The intensity of the 62-bp fragments generated from amplified cDNAs ranged from 14% to 19% at PCR cycles 22 through 30. Therefore, in heterozygotes the relative abundance of AE1 mRNA with the R664X mutation ranged from 28% to 38% of the total amount. This method also showed that in heterozygotes levels of AE1 mRNA relative to protein 4.1 were decreased to 55% of normal, in agreement with the estimation described above (A). The amounts of the R664X mutant (closed squares) and wild-type (open squares) mRNA in bone marrow cells from heterozygotes (He) and homozygotes (Ho) relative to that in normal (N) cells are shown (C).

• Supplemental Figure 2 -

  Fig. S2. Absence of N-glycan modification in bovine AE1. A, membranes from bovine, canine, and human red blood cells were incubated in the presence (PNGase) or absence (Mock) of peptide: N-glycosidase F and analyzed by SDS-PAGE, followed by staining with Coomassie brilliant blue. AE1 (band 3), protein 4.1 (a and b), and protein 4.2 along with positions of protein standards are indicated. Asterisks indicate deglycosylated polypeptides of canine and human AE1. B, in vitro translation in the presence of canine pancreatic microsomes of bovine AE1 wild type (bebWT) and a P661S mutant (bebP661S). P661S AE1 protein contained both N-glycosylated and non-glycosylated forms.