1. Volume 120, Issue 4, Pages 967-974 (March 2001)
A mutation of the Wilson disease protein, ATP7B, is degraded in the proteasomes and forms protein aggregates 
Masaru Harada, Shotaro Sakisaka, Kunihiko Terada, Rina Kimura, Takumi Kawaguchi, Hironori Koga, Motoaki Kim, Eitaro Taniguchi, Shinichiro Hanada, Tatsuo Suganuma, Koh Furuta, Toshihiro Sugiyama, Michio Sata 
Gastroenterology 
Volume 120, Issue 4, Pages (March 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Expression constructs used in the study. Some constructs contain an NH2-terminal GFP tag as indicated. Point mutated ATP7B cDNAs were produced by site-directed mutagenesis with the oligonucleotide primers. H1069Q is mutated in the SEHPL motif, highly conserved in heavy metal–transporting ATPases. N1270S is mutated in the hinge domain. Each constructed cDNA was inserted into the expression vectors for mammalian cells.
Gastroenterology  , DOI: ( /gast )
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3. Fig. 2
Confocal laser scanning microscopic images of (A–G) GFP-ATP7B–transfected or (H–J) GFP-N1270S–transfected Huh7 cells. (E–G) Some cells were treated with bathocuproine disulfonate. Cells were stained by anticalnexin (A), anti-GalT (B, E), anti–lamp 1 (C, F, I), anti–cathepsin D (D, G, J), and anti-ATP7B antibodies (H). GFP-ATP7B overlapped in its distribution with lamp 1, but not with calnexin, GalT, or cathepsin D. Bathocuproine disulfonate did not affect the distribution of GFP-ATP7B. The green fluorescence of GFP-N1270S was identical to the signals detected with anti-ATP7B antibody, and GFP-N1270S colocalized with lamp 1 but not with GalT or cathepsin D. Bar = 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Confocal laser scanning microscopic images of (A–F) GFP-H1069Q– or (G) H1069Q cDNA–transfected Huh7 cells. (B and C) GFP-H1069Q–transfected cells were labeled with anti-ATP7B antibody. GFP signals were observed throughout the cells, and they were not recognized by anti-ATP7B antibody. GFP signals aggregated around the nucleus in some cells, and they were labeled with anti-ATP7B antibody (D–F). H1069Q cDNA–transfected cells were stained by anti-ATP7B antibody (G). The signals were observed as a reticular pattern in the cytoplasm without nucleus. Bar = 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Confocal laser scanning microscopic images of GFP-H1069Q– or H1069Q cDNA–transfected Huh7 cells. GFP-H1069Q–transfected cells were treated with proteasome inhibitors, (A–C) lactacystine and (D–F) ALLN, for 24 hours and labeled with anti-ATP7B antibody (B, C, E, F). Perinuclear aggresomes were labeled with anti-ATP7B antibody. ALLN-treated H1069Q cDNA–transfected cells labeled with anti-ATP7B antibody revealed similar aggresomes (G). GFP signals were observed throughout the cells in bafilomycin A1-, a vacuolar H+-ATPase inhibitor, treated GFP-H1069Q–transfected cells similar to untreated cells (H). Cells treated with ALLN were labeled with anticalnexin (I), anti-GalT (J), and lamp 1 (K). Neither calnexin, GalT, nor lamp 1 colocalized with the aggresomes. Bar = 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

6. Fig. 5
Transmission electron micrograph of a GFP-H1069Q–transfected HEK293 cell. Aggresomes (arrow) composed of electron-dense materials located near the nucleus were observed. Bar = 1 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Confocal laser scanning microscopic images of (A–F, J–L) GFP-H1069Q–transfected Huh7 and (G–I, M–Q) HEK293 cells. IFs were labeled with antipancytokeratin (B, C, E, F) and antivimentin (H, I) antibodies in Huh7 and HEK293 cells, respectively. IFs extended into a cellular network in untreated GFP-H1069Q–transfected Huh7 cells (B, C), but they redistributed to surround the aggresomes in ALLN-treated Huh7 (E, F) and HEK293 (H, I) cells. MTOC was labeled with anti–γ-tubulin antibody (J–L). Aggresomes always localized at the MTOC in both Huh (J–L) and HEK293 (M–O) cells. The MT network was labeled with anti–α-tubulin antibody, and aggresome formation did not affect the MT network in HEK293 cells (P). MT disruption by nocodazole did not affect the aggresome formation in HEK293 cells (Q). Bar = 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions