Mic19 chchd3

The sample for the western blotting was prepared by scraping the cells in PBS. The protein extraction was done using RIPA lysis buffer. The amount of protein was determined using Bradford reagent and spectrophotometer for equal loading of a gel. The samples were prepared in Laemmli loading buffer. 10–20% precast Tricine gel (Invitrogen) was used (particularly for MIC13 as it is a small protein). The proteins were transferred onto nitrocellulose membrane and probed for various antibodies. Anti-HRP secondary antibodies were used. We used following antibodies for immunoblotting, MIC13 (Pineda, Berlin, polyclonal antibody raised in rabbit using following peptide CKAREYSKEGWEYVKARTK), MIC27/APOOL (Atlas Antibodies, HPA000612), MIC26/APOO (Thermo-Fisher, MA5-15493) MIC60/Mitofilin (Pineda, Berlin, polyclonal antibody raised in rabbit against human IMMT using the peptide CTDHPEIGEGKPTPALSEEAS), MIC10/MINOS1 (Pineda, Berlin, polyclonal antibody raised in rabbits against CQHDFQAPYLLHGKYVK), MIC25/CHCHD6 (cell signaling), VDAC (abcam), β-tubulin (Cell signaling), MIC19/CHCHD3 (abcam), ATP5L (proteintech), ATP5O (abcam), COXIV (abcam), NDUFB4 (abcam), UQCRC2 (abcam), MTND1 (abcam), TOM20 (Proteintech), TIM23 (BD biosciences), human TAZ1 (gift from Steve Claypool). Recording and visualization of chemiluminescent signals were done using VILBER LOURMAT Fusion SL (Peqlab).

FLAG, Actin and Mic23/ApoO antibodies were purchased from Sigma (St. Louis, Missouri USA), Tom20 and F1β from BD Transduction Laboratories (Franklin Lakes, New Jersey, USA), Mic60/Mitofilin, Mic19/CHCHD3 and Mic25/CHCHD6 from Abcam (Cambridge, UK), SDHA, Core1 and CoxI from Invitrogen/Thermo Fisher Scientific (Massachusetts, USA), Mic10/MINOS1, UQCRFS1 and CoxII from GeneTex (Irvine, California, USA), GAPDH, Tubulin, Tom40 and NDUFS1 from Santa Cruz (Dallas, Texas, USA), Hsp60 from Enzo Life Sciences (Farmingdale, New York, USA), and ArmC1 from Life Technologies (Carlsbad, California, USA). Sam50, Metaxin 1 and ArmC1 antibodies were raised in rabbits against the full-length 10xHis-tagged protein. Rat Tom70 antibody (crossreactive with human Tom70) was a kind gift from K. Mihara. Secondary anti-rabbit and anti-mouse antibodies coupled to Cy3 or Cy5 fluorophores were purchased from Dianova (Hamburg, Germany).