Reviewer's report 
 Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine Title: 
 2  16 July 2004 Version: Date: 
 Victor Ling Reviewer: 
Reviewer's report: 
General 
This study using human tissues has the potential to uncover novel aspects of ABCG5 and ABCG8 
biology. The prevailing wisdom is that G5 and G8 function as heterodimers to transport sterols in various tissues. The co-localization in vivo of these proteins have not been well-demonstrated. Understanding the mechanism of how these proteins interact and function is fundamental to 
understanding sterol homeostasis and sitosterolemia. An important possibilty raised by the present 
study is that G5 and/or G8 can act alone or with some other partner not yet identified. This is based on the observation that G5 and G8 have distribution patterns different from each other when their localization is determined by immunohistochemical staining. Some of the conclusions drawn in the 
paper is not stronly supported by the experimental data need further clarifications. Some queries that 
need to be addressed are noted below. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1) The authors should provide more detail on page 6 as to how the peptides used for raising 
antibodies to G5 and G8 were chosen. Since these are conserved proteins across species, it 
puzzling that the cross-reactivity is low in mice and not detectable in rats (Figure 1). Were the peptides designed to be human specific? If so, why is this an advantage? In the same vein, what evidence is there that these antisera do not cross-react with each other, that is, G5 antiserum does 
not detect G8 etc? The authors also need to assure the reader that the antisera do not cross react 
with other proteins in the western blots. Are we provided with a limited view of the gels or are the 
blots shown in Figure 1 represent entire gels. Do the gels include proteins with higher molecular 
weigth than 150 kD for example? These are important issues as any cross-reacting proteins can 
affect the interpretation of the apparent distribution patterns of these proteins. 
2)The conclusion that G5 and G8 co-localized with each other and function as heterodimers (bottom 
of page 10)is not as strongly supported by the data as it could be. The conclusion in Figure 2 that G5 
and G8 fractionate similarly in the sucrose gradients is generally correct but not precisely correct.In 
Figure 1A, G5 "peaked" in fraction 10 while G8 "peaked" in fraction 9 and, in Figure 1B, G5 peaked in fraction 11 and G8 in fraction 12. These data suggest that the highest concentrations of these proteins are actually in different membrane fractions. These data do not preclude that G5 and G8 
can form heterodimers but the data cannot be used in strong support that the majority of G5 and G8 
are in that form. Thus, statements such as those on bottom of page 10 need to be modified. While the immunohistochemical staining data are convincing that different staining patterns are observed for G5 and G8 ( as shown in Figure 3) evidence for the co-localization of G5 and G8 as would be 
predicted perhaps for the bile canalicular membranes is lacking and would be important to 
demonstrate convincingly. Merged images may allow for the detection of the membranes where G5 and G8 are co-localized. The discussion need to be revised in light of these comments. 
3))The blot for MDR1 in Figure 2 is poor. If C219 antibody is used it does not distiguish very well 
between MDR1 and MDR2/3. MDR1 is generally not highly expressed in liver membranes whereas 
MDR2/3 is expressed in the canalicular membranes at a relatively high level. Thus it is unwise to use 
MDR1 as a marker for the canalicular membrane as suggested at the bootom of page 12. It may be 
better to use BSEP ( bile salt export pump or sister of P-glycoprotein, ABCB11) as a marker. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
None 
Reviewer's report 
 Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine Title: 
 2  16 July 2004 Version: Date: 
 Victor Ling Reviewer: 
Reviewer's report: 
General 
This study using human tissues has the potential to uncover novel aspects of ABCG5 and ABCG8 
biology. The prevailing wisdom is that G5 and G8 function as heterodimers to transport sterols in various tissues. The co-localization in vivo of these proteins have not been well-demonstrated. Understanding the mechanism of how these proteins interact and function is fundamental to 
understanding sterol homeostasis and sitosterolemia. An important possibilty raised by the present 
study is that G5 and/or G8 can act alone or with some other partner not yet identified. This is based on the observation that G5 and G8 have distribution patterns different from each other when their localization is determined by immunohistochemical staining. Some of the conclusions drawn in the 
paper is not stronly supported by the experimental data need further clarifications. Some queries that 
need to be addressed are noted below. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1) The authors should provide more detail on page 6 as to how the peptides used for raising 
antibodies to G5 and G8 were chosen. Since these are conserved proteins across species, it 
puzzling that the cross-reactivity is low in mice and not detectable in rats (Figure 1). Were the peptides designed to be human specific? If so, why is this an advantage? In the same vein, what evidence is there that these antisera do not cross-react with each other, that is, G5 antiserum does 
not detect G8 etc? The authors also need to assure the reader that the antisera do not cross react 
with other proteins in the western blots. Are we provided with a limited view of the gels or are the 
blots shown in Figure 1 represent entire gels. Do the gels include proteins with higher molecular 
weigth than 150 kD for example? These are important issues as any cross-reacting proteins can 
affect the interpretation of the apparent distribution patterns of these proteins. 
2)The conclusion that G5 and G8 co-localized with each other and function as heterodimers (bottom 
of page 10)is not as strongly supported by the data as it could be. The conclusion in Figure 2 that G5 
and G8 fractionate similarly in the sucrose gradients is generally correct but not precisely correct.In 
Figure 1A, G5 "peaked" in fraction 10 while G8 "peaked" in fraction 9 and, in Figure 1B, G5 peaked in fraction 11 and G8 in fraction 12. These data suggest that the highest concentrations of these proteins are actually in different membrane fractions. These data do not preclude that G5 and G8 
can form heterodimers but the data cannot be used in strong support that the majority of G5 and G8 
are in that form. Thus, statements such as those on bottom of page 10 need to be modified. While the immunohistochemical staining data are convincing that different staining patterns are observed for G5 and G8 ( as shown in Figure 3) evidence for the co-localization of G5 and G8 as would be 
predicted perhaps for the bile canalicular membranes is lacking and would be important to 
demonstrate convincingly. Merged images may allow for the detection of the membranes where G5 and G8 are co-localized. The discussion need to be revised in light of these comments. 
3))The blot for MDR1 in Figure 2 is poor. If C219 antibody is used it does not distiguish very well 
between MDR1 and MDR2/3. MDR1 is generally not highly expressed in liver membranes whereas 
MDR2/3 is expressed in the canalicular membranes at a relatively high level. Thus it is unwise to use 
MDR1 as a marker for the canalicular membrane as suggested at the bootom of page 12. It may be 
better to use BSEP ( bile salt export pump or sister of P-glycoprotein, ABCB11) as a marker. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
None 
Please review this paper.
user
Reviewer's report 
 Localization of ABCG5 and ABCG8 proteins in human liver, gall bladder and intestine Title: 
 2  16 July 2004 Version: Date: 
 Victor Ling Reviewer: 
Reviewer's report: 
General 
This study using human tissues has the potential to uncover novel aspects of ABCG5 and ABCG8 
biology. The prevailing wisdom is that G5 and G8 function as heterodimers to transport sterols in various tissues. The co-localization in vivo of these proteins have not been well-demonstrated. Understanding the mechanism of how these proteins interact and function is fundamental to 
understanding sterol homeostasis and sitosterolemia. An important possibilty raised by the present 
study is that G5 and/or G8 can act alone or with some other partner not yet identified. This is based on the observation that G5 and G8 have distribution patterns different from each other when their localization is determined by immunohistochemical staining. Some of the conclusions drawn in the 
paper is not stronly supported by the experimental data need further clarifications. Some queries that 
need to be addressed are noted below. 
------------------------------------------------------------------------------- 
Major Compulsory Revisions (that the author must respond to before a decision on publication can 
be reached) 
1) The authors should provide more detail on page 6 as to how the peptides used for raising 
antibodies to G5 and G8 were chosen. Since these are conserved proteins across species, it 
puzzling that the cross-reactivity is low in mice and not detectable in rats (Figure 1). Were the peptides designed to be human specific? If so, why is this an advantage? In the same vein, what evidence is there that these antisera do not cross-react with each other, that is, G5 antiserum does 
not detect G8 etc? The authors also need to assure the reader that the antisera do not cross react 
with other proteins in the western blots. Are we provided with a limited view of the gels or are the 
blots shown in Figure 1 represent entire gels. Do the gels include proteins with higher molecular 
weigth than 150 kD for example? These are important issues as any cross-reacting proteins can 
affect the interpretation of the apparent distribution patterns of these proteins. 
2)The conclusion that G5 and G8 co-localized with each other and function as heterodimers (bottom 
of page 10)is not as strongly supported by the data as it could be. The conclusion in Figure 2 that G5 
and G8 fractionate similarly in the sucrose gradients is generally correct but not precisely correct.In 
Figure 1A, G5 "peaked" in fraction 10 while G8 "peaked" in fraction 9 and, in Figure 1B, G5 peaked in fraction 11 and G8 in fraction 12. These data suggest that the highest concentrations of these proteins are actually in different membrane fractions. These data do not preclude that G5 and G8 
can form heterodimers but the data cannot be used in strong support that the majority of G5 and G8 
are in that form. Thus, statements such as those on bottom of page 10 need to be modified. While the immunohistochemical staining data are convincing that different staining patterns are observed for G5 and G8 ( as shown in Figure 3) evidence for the co-localization of G5 and G8 as would be 
predicted perhaps for the bile canalicular membranes is lacking and would be important to 
demonstrate convincingly. Merged images may allow for the detection of the membranes where G5 and G8 are co-localized. The discussion need to be revised in light of these comments. 
3))The blot for MDR1 in Figure 2 is poor. If C219 antibody is used it does not distiguish very well 
between MDR1 and MDR2/3. MDR1 is generally not highly expressed in liver membranes whereas 
MDR2/3 is expressed in the canalicular membranes at a relatively high level. Thus it is unwise to use 
MDR1 as a marker for the canalicular membrane as suggested at the bootom of page 12. It may be 
better to use BSEP ( bile salt export pump or sister of P-glycoprotein, ABCB11) as a marker. 
------------------------------------------------------------------------------- 
Minor Essential Revisions (such as missing labels on figures, or the wrong use of a term, which the author can be trusted to correct) 
------------------------------------------------------------------------------- 
Discretionary Revisions (which the author can choose to ignore) 
 Unable to decide on acceptance or rejection until the authors have responded to the What next?: 
major compulsory revisions 
 An article of importance in its field Level of interest: 
 Acceptable Quality of written English: 
 No Statistical review: 
Declaration of competing interests: 
None 
assistant