A merged image reveals no colocalization of the MTS-CFP and RFP signals. in SN, comparable to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention. (cyt and 1 gene (MERLWGLFQRAQQLSPRSSQTVYQRVEG) was subcloned by PCR from a expression vector (Genecopoeia, Germantown, MD). Primers TFRmito-F (5-CATGGTCGACGCCACCATGGAGCGGCTTTGGGGTCTA-3) and TFRmito-R (5-CATGGGTACCAACCCTTTCCGGGGGCCTTCCA-3) were used to amplify the MTS and generate unique Sal I and Kpn I restriction sites around the 5 and 3 ends respectively of the Iloprost PCR product. The PCR product was subcloned in frame into the multiple cloning site of the pDsRED-monomer (Clontech, Mountain View, CA) to generate the TfR2 MTS RFP (reddish fluorescent protein) vector. Transfection of HEK 293T cells Twenty-four hours prior to transfection, 2.25105 HEK 293T cells were plated in each well of a 6 well dish. Cells were incubated for 24 h at 37 C in a 5% CO2 atmosphere. Prior to transfection, DNA (4 g cyan fluorescent protein (CFP) construct + 4 g of RFP construct) and Lipofectamine 2000 reagent (Invitrogen, Temecula,CA) were combined and incubated according to the manufacturer’s recommendations. DNA/Lipofectamine 2000 mixtures were applied to cells made up of 5.0 ml of serum-free DMEM. The DNA combination was removed after 6 h of incubation and was replaced with 5.0 ml of DMEM containing 5% fetal calf serum. Transfected cells continued to grow for an additional 18 h prior to confocal microscopy analysis. TfR2 silencing Five clones of TfR2 silencing shRNA (Mission? shRNA, Sigma, St. Louis, MO) were purchased from Sigma. The silencing ability of each clone was assessed 24 Iloprost h after transfection by western blot. The most effective clone (TRCN0000063628) C which reduced TfR2 expression by 90% C was used for all the experiments. RT-PCR Human tissue RNA was obtained from Ambion Inc. (Austin, TX). RT-PCR was performed using 2 g total RNA and the SuperScript RT-PCR system (Invitrogen, Carlsbad, Rabbit Polyclonal to GA45G CA). TFR2-specific PCR was performed using two unique primer units (Kawabata et al., 1999). Set A: For: 5-GTGGTCAGTGAGGATGTCA-3; Rev: 5-CGTGGTCCA-GCTTCTGGCGGGAG-3. Set B: For: 5-ACGTCTCTGGCATCCTTCC-3; Rev: 5-CATCGACCCAGTGCAGGGTG-3. Treatment of TfR2 overexpressing cells with Alexa-conjugated transferrin 100 g of holo-transferrin (Sigma) was labeled with the Alexa488 protein labeling kit (Molecular Probes, Eugene, OR) according to the manufacturer’s training. HEK 293 cells overexpressing transferrin receptor 2 and mitochondrial targeted CFP (Invitrogen, Temecula, CA), were kept in serum-free medium 30 min at 37 C, 5% CO2, to allow cells to unload endogenous transferrin from endocytic vesicles. Then cells were incubated for 60 min at 4 C with labeled transferrin (3.2 nM). Cells were washed 3 times with ice-cold HBSS and then incubated again in standard cell culture medium. After 90 min, cells were stained with the potentiometric mitochondrial dye tetramethylrhodamine methyl ester TMRM (20 nM, Molecular Probes, Eugene, OR) and analyzed with an inverted laser scanning confocal microscope (Fluoview 1000, Olympus). Release of ferrous iron (Fe2+) from transferrin Release of ferrous iron was measured as previously explained (Kojima and Bates, 1979), by monitoring the absorbance of the Fe2+BPS complex (bathophenanthroline disulfonate, Sigma, St. Louis, MO) at 538 nm with a Spectramax Plus spectrophotometer (Molecular Devices, Sunnyvale, CA). BPS chelates ferrous iron exclusively and it Iloprost was used 1 mM final. 1 mg of Tf of NEM-Tf was used for each reaction. The reactions were carried out in 100 mM TrisHCl pH 7.4. Xanthine was used 3 mM final, xanthine oxidase was used 0.1 U/mL. Oxidized glutathione (Sigma, St. Louis, MO) was prepared immediately before adding it to the reaction and used 7.5 mM. For all the solutions, the pH was adjusted to 7.4. Absorbance was measured after 20 min of incubation at room temperature. At the end of each experiment, concentrated HCl was added to induce total iron release; the absorbance observed after HCl mediated ferrous release was set as 100% and used to normalize the values obtained in each experiment. Where indicated, transferrin was alkylated before the experiment with 100 mM N-ethylmaleimide (NEM) in 50 mM TrisHCl pH 6.8, room heat. Alkylation with NEM did not result in any Iloprost iron release (data not shown). Unreacted NEM was removed by microfiltration (Microcon.