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Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

Bottom Line: A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins.These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae.They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

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Related in: MedlinePlus

When transiently co-expressed, Cav1 with different tags can form hetero-oligomers. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for myc (red) or GFP (green). The signal for the Cav1-myc channel was deliberately overexposed to allow for detection of Cav1-myc signal in the channel corresponding to the co-transfected cells.
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fig06: When transiently co-expressed, Cav1 with different tags can form hetero-oligomers. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for myc (red) or GFP (green). The signal for the Cav1-myc channel was deliberately overexposed to allow for detection of Cav1-myc signal in the channel corresponding to the co-transfected cells.

Mentions: To distinguish between these possibilities, we performed two independent experiments. In the first experiment, we transiently co-transfected COS-7 cells with Cav1-GFP and Cav1-myc. As Cav1-GFP is simultaneously expressed with Cav1-myc, we predicted that they should form hybrid oligomers if they are capable of interacting with each other, and that the hybrid oligomer should have a molecular weight intermediate between that of pure Cav1-GFP and Cav1-myc oligomers. Western blot analysis of BN-PAGE gels confirmed this prediction. A hybrid oligomer could be detected with both GFP and myc antibodies in co-transfected cells, and this band was positioned between the pure Cav1-GFP and Cav1-myc oligomer bands (Figure 6). This result demonstrates that the overexpressed Cav1-GFP is capable of oligomerizing with a variant of Cav1 containing only a small epitope tag. However, this experiment does not directly demonstrate whether the Cav1-GFP is capable of oligomerizing with endogenous Cav1 and Cav2.


Tagging strategies strongly affect the fate of overexpressed caveolin-1.

Han B, Tiwari A, Kenworthy AK - Traffic (2015)

When transiently co-expressed, Cav1 with different tags can form hetero-oligomers. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for myc (red) or GFP (green). The signal for the Cav1-myc channel was deliberately overexposed to allow for detection of Cav1-myc signal in the channel corresponding to the co-transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440517&req=5

fig06: When transiently co-expressed, Cav1 with different tags can form hetero-oligomers. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for myc (red) or GFP (green). The signal for the Cav1-myc channel was deliberately overexposed to allow for detection of Cav1-myc signal in the channel corresponding to the co-transfected cells.
Mentions: To distinguish between these possibilities, we performed two independent experiments. In the first experiment, we transiently co-transfected COS-7 cells with Cav1-GFP and Cav1-myc. As Cav1-GFP is simultaneously expressed with Cav1-myc, we predicted that they should form hybrid oligomers if they are capable of interacting with each other, and that the hybrid oligomer should have a molecular weight intermediate between that of pure Cav1-GFP and Cav1-myc oligomers. Western blot analysis of BN-PAGE gels confirmed this prediction. A hybrid oligomer could be detected with both GFP and myc antibodies in co-transfected cells, and this band was positioned between the pure Cav1-GFP and Cav1-myc oligomer bands (Figure 6). This result demonstrates that the overexpressed Cav1-GFP is capable of oligomerizing with a variant of Cav1 containing only a small epitope tag. However, this experiment does not directly demonstrate whether the Cav1-GFP is capable of oligomerizing with endogenous Cav1 and Cav2.

Bottom Line: A significant amount of our current knowledge about caveolins and caveolae is derived from studies of transiently overexpressed, C-terminally tagged caveolin proteins.These findings suggest that differences in tagging strategies may be a source of variation in previously published studies of Cav1 and that overexpressed Cav1 may exert functional effects outside of caveolae.They also highlight the need for a critical re-evaluation of current knowledge based on transient overexpression of tagged Cav1.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN, USA.

Show MeSH
Related in: MedlinePlus