Anti mtco1

BN-PAGE was carried out as detailed^{61 (link)}. Samples containing 15 µg of protein were separated on 3–12% Bis–Tris Novex NativePAGE gel (Life Technologies). The relative levels of the assembled respiratory complexes I-V were assessed by western blot with following antibodies: anti-ND1 (Thermo Fisher, 19703-1-AP), anti-SDHA (Cell signaling, 5839), anti-MTCO1 (Thermo Fisher, 459600), anti-ATP5a (Abcam, 11448) and anti-Cytb (Abclonal, A17966). Horseradish-peroxidase-conjugated anti-rabbit IgG (Sigma, A6154) and anti-mouse IgG (Sigma, A4416) antibodies were used as secondary antibodies and protein signals were detected using the ECL system. Bis–Tris Novex NativePAGE (3–12%) gels were stained using Coomassie protein staining solution (50% methanol, 7% acetic acid and 0.1% Coomassie Brilliant Blue R stain).

Ovaries from fully engorged I. ricinus females were dissected under DEPC-treated PBS. Three individual ovaries were used for RNA extraction and three individual ovaries were homogenized and used for protein solubilization as described above. Protein homogenates of tick ovarian tissue or embryonic cell line were adjusted to the equal protein load and separated by reducing SDS-PAGE in 4–20% Stain-free gels (BioRad: 4568095). Protein separation was visualized in the stain-free mode and proteins were transferred to 0.2µm pore PVDF membrane (BioRad: 1704272) for Western blotting. The membrane was blocked in non-fat milk suspended in PBS-Tween for 1 hour at room temperature. Primary antibodies anti-ATP5A (Abcam: ab14748) and anti-MTCO1(Thermo Fisher Scientific: 459600) were diluted in PBS-Tween 1:500 and 1:1000, respectively, and incubated with the membrane overnight. Immunodetection was performed by anti-mouse peroxidase-conjugated immunoglobulin (Sigma-Aldrich: A9044), diluted 1: 10 000 in PBS-Tween, and the signal was visualized by Clarity™ Western ECL substrate (BioRad: 170-5061) using ChemiDoc (BioRad).

Electrophoresed gels were blotted onto polyvinylidene fluoride (PVDF) membranes using an iBlot transfer system (Invitrogen) according to the manufacturer’s instructions. Blotted PVDF membranes were blocked at room temperature for 30 min. Primary antibody probing was performed at room temperature for 90 min. Secondary antibody probing was performed with chromogenic antibody detection kit (WesternBreeze; Invitrogen) according to the manufacturer’s instructions. Primary antibodies used for SDS-PAGE were as follows: 0.5 μg/mL anti-porin (Molecular Probes), 2.5 μg/mL anti-MT-CO1 (Molecular Probes), 2.5 μg/mL anti-MT-CO2 (Molecular Probes), 2.5 μg/mL anti-COX4 (Molecular Probes), 2.5 μg/mL anti-COX5B (Molecular Probes). Primary antibodies used for BN-PAGE were as follows: 0.5 μg/mL anti-NDUFA9 for CI (Molecular Probes), 0.5 μg/mL anti-SDHA for CII (Molecular Probes), 0.5 μg/mL anti-UQCRC2 for CIII (Molecular Probes), 2.5 μg/mL anti-MT-CO1 for CIV (Molecular Probes), 0.5 μg/mL anti-ATP5B for CV (Molecular Probes).