Mouse anti ng

Western blot analyses were performed with isolated mitochondria. Fifty micrograms of isolated mitochondria was preheated on 95°C for 5 min in 1× SDS loading buffer and separated on a 12% SDS gel. After transfer to polyvinylidene difluoride membranes, membranes were incubated with the following primary antibodies in 5% milk and tris-buffered saline: mouse anti-NG (1:1000; Chromotek GmbH), rabbit anti-aconitase1 (1:1000), and rabbit anti-Atp6 (1:1000). Quantitative real-time PCR experiments to determine mtDNA levels were performed as described previously (51 (link)). For the petite analysis, cells were grown overnight at 30°C, then freshly diluted to an OD₆₀₀ = 0.2, and grown for another 3 hours. A total of 200 cells were plated onto YPG plates containing 0.1% glucose. Plates were incubated for 3 days at 30°C. Only cells proficient in respiratory growth are able to continue growth after all glucose has been consumed.

To isolate flagella for protein analysis, we followed a protocol described by Witman (1986) (link). In brief, cells were concentrated and washed in 10 mM HEPES, pH 7.4, resuspended in HMS at 4°C (10 mM HEPES, 5 mM MgSO₄, and 4% sucrose) and deflagellated by adding dibucaine to a final concentration of 4.17 mM (Sigma-Aldrich) and repeated pipetting. Flagella were separated from cell bodies by centrifugation, collected from the supernatant by centrifugation, and resuspended in HMEK (30 mM HEPES, 5 mM MgSO₄, 25 mM KCl, and 0.5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid [EGTA]) with protease inhibitor cocktail (Sigma-Aldrich). For Western blotting, SDS–PAGE sample buffer was added to flagella samples and samples were incubated at 85°C for 10 min. The following primary antibodies were used: mouse anti-IC2 (1:1000; King and Witman, 1990 (link)), mouse anti-IFT81 (1:1000; Cole et al., 1998 (link)), rabbit anti-ODA16 (1:200; Ahmed and Mitchell, 2005 (link)), and mouse anti-NG (1:1500; Chromotek). Western blots were developed using anti-mouse and anti-rabbit immunoglobulins G conjugated to horseradish peroxidase (Invitrogen) and chemilu­minescence substrate (Michigan Diagnostics). Images were captured using a BioRad Gel Doc imaging system.