Subramanian VS , Nabokina SM , Lin-Moshier Y , Marchant JS , Said HM .

R01 DK058057 NIDDK NIH HHS , R01 AA018071 NIAAA NIH HHS , AA 18071 NIH, R37 DK056061 NIDDK NIH HHS , R01 GM088790 NIGMS NIH HHS , R01 DK056061 NIDDK NIH HHS

	Figure 2. Expression of MTPPT in the mouse liver and HepG2 cells at the mRNA and protein levels.A) Total RNA was isolated and quantitative real-time PCR was performed as described in “Methods”. Data (mean ± SE) presented are from four independent mice normalized relative to ARPO or multiple samples from HepG2 cells were normalized relative to β-actin. B) Western blot analysis was performed with the use of isolated mouse liver or HepG2 cells mitochondria (100 µg) and specific anti-MTPPT polyclonal antibodies as described in “Methods”. Data from a representative experiment involving two separate mitochondria preparations from two mice or HepG2 cells are shown.
	Figure 3. Cellular distribution of hMTPPT-GFP or GFP-hMTPPT in HepG2 cells and X. Oocytes.A) Predicted 6 transmembrane domains of hMTPPT showing the location of three-fold tandem repeated domains and both NH2 and COOH-terminals oriented towards the cytosol. B) Lateral (xy) confocal images of hMTPPT-GFP or GFP-hMTPPT co-transfected with DsRed-Mito (mitochondrial marker) and their overlay in HepG2 cells. C) Resolution of hMTPPT-GFP expression in a Xenopus oocyte. Top, axial image (‘xz’, blue) of a cortical band of hMTPPT-GFP expression. Individual lateral (‘xy’, red) images are displayed as image planes taken at successive 1 µm intervals into the oocyte cortex. D) Lateral (xy) confocal images of hMTPPT-GFP co-transfected with DsRed-Mito and their overlay in Xenopus oocyte. E) Western blot was performed on two individual hMTPPT-GFP expressing oocytes and probed with mouse anti-GFP (1∶1000) and goat anti-mouse antibodies (1∶5000).
	Figure 4. Systematic truncation of the hMTPPT polypeptide and their expression pattern in HepG2 cells.Sub cellular expression of indicated (five) hMTPPT-GFP truncated constructs in HepG2 cells. Lateral (xy) confocal images of different truncated constructs were imaged after 24–48 hrs of transient transfection.
	Figure 5. Systematic truncation of the hMTPPT polypeptide and their expression pattern in HepG2 cells.Sub cellular expression of indicated (four) hMTPPT-GFP truncated constructs in HepG2 cells. Lateral (xy) confocal images of different truncated constructs were imaged after 24–48 hrs of transient transfection.
	Figure 6. Cellular expression of hMTPPT clinical mutants and their function in stable HepG2 cells.A) The Raswin program which predicts the location of the clinical mutants (G125S and G177A) in the hMTPPT polypeptide. B) Lateral (xy) scan of hMTPPT-GFP (WT) and clinical mutants were co-transfected with DsRed-mito and their overlay in stable HepG2 cell line. C) 3H-TPP (0.38 µM)) uptake by stable HepG2 cells expressing GFP (vector alone), hMTPPT-GFP (WT) and clinical mutants. Initial-rate of 3H-TPP uptake was performed on mitochondria isolated from HepG2 cells. The uptake was linear up to 10 min (data not shown). D) Western blot analyses were performed on isolated mitochondria from hMTPPT (WT) and clinical mutants (60 µg protein) as described in “Methods”. Blots were incubated with mouse monoclonal GFP antibodies. Samples were normalized relative to pyruvate dehydrogenase (PDH) protein expression. Data are mean ± SE from at least three different samples from three different batches of cells. * p<0.01,** p<0.02,*** p<0.04.
	Figure 1. Characteristics of the TPP uptake process by the isolated mouse liver mitochondria. A) Uptake as a function of time. Mitochondria were incubated in uptake buffer, pH 7.4, at 37°C. 3H-TPP (0.38 µM) was added to the incubation medium at the start of uptake. Data are mean ± SE of 3–4 separate uptake determinations. B) Effect of incubation buffer pH on the initial-rate of 3H -TPP uptake. Mitochondria were incubated in uptake buffer of varying pH at 37°C. 3H-TPP (0.38 µM) was added to the incubation buffer at the onset of a 2 min of incubation time (i.e., initial-rate). Data are mean ± SE of 3–4 separate uptake determinations. C) Uptake of 3H-TPP as a function of substrate concentration. Mitochondria were incubated in uptake buffer, pH 7.4, at 37°C in the presence of different concentrations of TPP. Uptake was measured after 2 min incubation (i.e., initial-rate). Uptake by the carrier-mediated system was calculated as described in “Methods”. Data are mean ± SE of 3–4 separate uptake determinations. D) Effect of structural analogs of TPP on the initial-rate of uptake. Mitochondria were incubated in uptake buffer, pH 7.4, at 37°C in the presence of 3H-TPP (0.38 µM) and unlabeled TPP, TMP, thiamin, and biotin (120 µM for all). Uptake was measured after 2 min incubation (i.e., initial-rate). Data are mean ± SE of 5–12 separate uptake determinations. *p<0.01.

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