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Blood, Vol. 112, Issue 5, 2055-2061, September 1, 2008
A novel loss-of-function mutation in the proton-coupled folate transporter from a patient with hereditary folate malabsorption reveals that Arg 113 is crucial for function
Blood Lasry et al.
112: 2055
Supplemental materials for: Lasry et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 72.4 KB)
- Table S1. Summary of genomic PCR primers used and the position of their PCR products (PDF, 20.5 KB)
- Table S2. Summary of RT-PCR primers used and the cDNA position of the PCR products (PDF, 19.4 KB)
- Table S3. Folic acid growth requirement of wt- and mutant R113C PCFT transfectants (PDF, 16.7 KB)
- Table S4. Flow cytometric values of cellular F-MTX staining and its displacement by MTX (PDF, 17.8 KB)
- Figure S1. Topology and localization of HFM mutations in the human PCFT (JPG, 72.5 KB)
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The 337C>T nucleotide mutation resulted in a single amino acid substitution, R113C, that mapped to a highly conserved region residing in cytoplasmic loop 1. Based on the published putative TM organization of the human PCFT in the plasma membrane,1 R113 maps to the first cytoplasmic loop connecting TM2 and TM3. The branches attached to N58 denote the N-linked glycosylation of PCFT.
- Figure S2. PCFT mRNA and protein expression in stable transfectants (JPG, 113 KB)
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Total RNA was isolated from stable transfectants of the wt- and mutant PCFT and converted to cDNA. Then, PCFT transcript levels were determined by semi-quantitative RT-PCR using various cDNA dilutions (Panel A, section a); hamster β-actin cDNA titration was used as an internal control (Panel A, section b). Histograms of mean cellular protein levels of the various Myc-tagged PCFTs were determined by immunofluorescence labeling and flow cytometric analysis in the different stable transfectants. Individual flow cytometric tracings of Myc-tagged PCFT levels are in the various transfectants are depicted as insets above the individual histograms (Panel B). The detailed methodology for the quantification of cellular Myc-tagged PCFT levels is given above in Supplemental Materials and Methods.
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