blot : myc grim-19 com ii myc bn-page sds-page grim-19 grim19 y143a grim-19 y143d grim-19 g139v...
Post on 17-Jan-2016
226 Views
Preview:
TRANSCRIPT
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.
top related