Pgem 4z vector

The OVA plasmid used for this study and generation of mRNA were described previously9 (link). For the hemagglutinin (HA), the coding sequence from influenza A virus subtype H1N1 (GenBank Accession Number: CY045764) was generated by gene synthesis (Takara) and cloned into the HindIII and BamHI sites of pGEM-4Z vector (Promega). The resultant plasmid was then linearized with BamHI and purified with QIAquick PCR Purification Kit (Qiagen). mRNA for HA was synthesized by in vitro transcription with the above linearized template using mMessage mMachine T7 Ultra Kit (Ambion) and purified using RNeasy Mini/Midi Kits (Qiagen). The integrity of the mRNA was analyzed by agarose gel electrophoresis on denaturing conditions.
To load NIH3T3 cells with α-GalCer, the cells were cultured for 48 h in the presence of 500 ng/mL α-GalCer and then washed three times before electroporation. α-GalCer-loaded NIH3T3 cells (NIH3T3/Gal) were transfected with antigen mRNA together with murine CD1d mRNA using a square pulse electroporator (BTX ECM630, Harvard Apparatus) as described41 (link). For transfection, electroporation was carried out using 5 μg of mCD1d mRNA and 10 μg of OVA or 10 μg of HA mRNA by a single 500 V/3 msec pulse. Immediately after electroporation, the cells were transferred to culture medium in the presence of 500 ng/ml of α-GalCer.

cDNA encoding NDUFA9 was cloned into the pGEM4Z vector (Promega, Madison, WI, USA) and protein translated using the TnT Coupled Reticulocyte Lysate System (Promega) in the presence of [35S]-methionine/cysteine. Translation products were incubated with freshly isolated mitochondria in 250 mM sucrose, 80 mM potassium acetate, 5 mM magnesium acetate, 10 mM sodium succinate, 1 mM dithiothreitol, 5 mM ATP and 20 mM HEPES pH 7.4 at 37 °C for the times indicated. Dissipation of the mitochondrial membrane potential (Δψ_m) was performed in the presence of 10 μM FCCP (with no ATP or sodium succinate). Samples subjected to protease treatment were incubated on ice for 10 min with 100 μg/mL proteinase K (Sigma) before treatment with 1 mM PMSF for 10 min. 40 μg of each sample was resolved on either SDS–PAGE or BN–PAGE as described, with proteins transferred to PVDF membranes before exposure to storage phosphor screens (GE Healthcare) and detection using a Typhoon Laser Scanner (GE Healthcare). Protein band intensities were calculated using ImageJ (NIH) software from at least three independent experiments, normalised to the maximum amount of imported NDUFA9 after 60 min in controls, and converted to % of control levels at 60 min.