Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells.
Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.
Department of Histology and Oral Histology, School of Dentistry, The University of Tokushima, 3-18-15 Kuramoto-cho, 770-8504 Tokushima, Japan.
Eiko
E
Sasaki
Yoshifumi
Y
Okamoto
Kaya
K
Yoshida
Hirohiko
H
Okamura
Katsuhide
K
Shimizu
Fumio
F
Nasu
Hiroyuki
H
Morimoto
Tatsuji
T
Haneji
D041721:3T3-L1 Cells / Q000737:chemistry / Q000648:ultrastructure; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D051379:Mice; D008928:Mitochondria / Q000737:chemistry*; D024101:Mitochondrial Proteins / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2; D019809:Streptavidin; D051028:bcl-2-Associated X Protein
C494827:Bax protein, mouse; D007202:Indicators and Reagents; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D019809:Streptavidin