Blot :

myc

GRIM-19

com II

myc

BN-PAGE

SDS-PAGE

GRIM-1

9

GRIM19

Y14

3A

GRIM-1

9 Y14

3D

GRIM-1

9 G13

9VGRIM

-19

G139R

Supplementary Fig. 1

GRIM19 mutants (G139V, G139R, Y143D and Y143A) were transfected into 293T cells. Blue-Native PAGE was used to resolve mitochondrial respiratory chain complexes from these cells. Anti-myc antibody was used to detect transfected GRIM-19 and mutants. Blue-Native Western blot was used to check the amount of exogenous proteins inserted into mitochondrial complex I. Normal SDS-PAGE/Western blot was also conducted to check the total amount of exogenous protein present in mitochondria. Mitochondrial complex II was detected by anti-complex II 70 kDa subunit antibody.

Blot : HA

GRIM-19

comII 70 kDa subunit

HA

BN PAGE

SDS PAGE

GRIM

-19-

HANDUFA

9 1-

100-

HA

NDUFA9

1-80

-HA

NDUFA9

1-60

-HA

NDUFS3

1-10

0-HA

NDUFS3

1-80

-HA

NDUFS3

1-60

-HA

Supplementary Fig. 2

NDUFA9 mutants (1-100 and 1-80) and NDUFS3 mutant (1-100) can be assembled into mitochondrial complex I. WT-GRIM-19, NDUFA9 mutants (1-100, 1-80 and 1-60), and NDUFS3 mutants (1-100, 1-80 and 1-60) were transfected into 293T cells. The same procedure was conducted as in Supplementary Fig 1.

Complex I

IP H

A

DN-GRIM-19

Supplementary Fig. 3

Mitochondrial complex I was immunoprecipated by complex I capture kit (MitoSciences (Eugene, OR, USA) according to manufacturer’s instructions. DN-GRIM-19-HA incorporated into complex I was immunoprecipated by HA-conjugated agarose beads. The immunoprecipated protein was eluted from the beads and subjected to SDS-PAGE. The gel was visualized by silver stain.