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. Author manuscript; available in PMC: 2020 Jul 1.
Published in final edited form as: Hum Mutat. 2019 Apr 13;40(7):893–898. doi: 10.1002/humu.23753

Figure 2. Characterizing the effect of the TIMMDC1 p.(Arg225*) variant on complex I assembly.

Figure 2.

a. BN-PAGE western blot of fibroblast protein showed a mild reduction of complex I (detected using antibody against NDUFA9) in the patient (P) relative to control (C). Levels of complex III (UQCRC1) and complex IV (COXIV) in the patient were comparable to control. Complex II (SDHA) is indicative of loading.

b. SDS-PAGE western blot of protein from fibroblasts showing protein levels of complex I subunits (MT-ND1, NDUFS2, NDUFS3, NDUFS5, NDUFA9 and NDUFB6) and complex I assembly factors (ACAD9, ECSIT, NDUFAF1 and NDUFAF4) in the patient and a control; subunits NDUFS5 and MT-ND1 were most reduced in the patient relative to control. An antibody targeting the C-terminus of TIMMDC1 (amino acids 212–284) was used. Complex II subunit SDHA was used as a loading control.

c. BN-PAGE western blot of control and TIMMDC1 knockout HEK293T cells expressing wild-type or TIMMDC1 p.(Arg225*) protein with C-terminal FLAG-tag. Overexpression of the wild-type and TIMMDC1 p.(Arg225*) mutant protein restored complex I assembly equally in TIMMDC1 knockout cells. An antibody against NDUFA9 was used to detect complex I. SDS-PAGE western blot using an anti-FLAG antibody was used to confirm expression of the FLAG-tagged TIMMDC1 proteins. Expected size of the FLAG-tagged wild-type and TIMMDC1 p.(Arg225*) protein is 33.2 kDa and 26.1 kDa, respectively. Complex II subunit SDHA is indicative of loading.