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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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The oligomerization state of overexpressed Cav1 varies as a function of its tag. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for Cav1 (red) and either GFP, mCherry or myc (green). A) Cells were either left untransfected (‘control’) or transfected with EGFP, Cav1-GFP or P132L-GFP. B) As in (A) except cells were transfected with the indicated mCherry constructs. C) As in (A) except cells were transfected with Cav1-myc or P132L-myc. Figures are representative of two independent experiments. Red arrows indicate the high molecular weight band positive for both tag antibodies and Cav1 antibodies (h1-97 or 2297). Black arrows indicate the high molecular weight band only positive for Cav1 antibodies (h1-97 or 2297). Green arrows indicate the low molecular weight bands only positive for FP tag antibodies.
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fig03: The oligomerization state of overexpressed Cav1 varies as a function of its tag. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for Cav1 (red) and either GFP, mCherry or myc (green). A) Cells were either left untransfected (‘control’) or transfected with EGFP, Cav1-GFP or P132L-GFP. B) As in (A) except cells were transfected with the indicated mCherry constructs. C) As in (A) except cells were transfected with Cav1-myc or P132L-myc. Figures are representative of two independent experiments. Red arrows indicate the high molecular weight band positive for both tag antibodies and Cav1 antibodies (h1-97 or 2297). Black arrows indicate the high molecular weight band only positive for Cav1 antibodies (h1-97 or 2297). Green arrows indicate the low molecular weight bands only positive for FP tag antibodies.

Mentions: We next tested for possible defects in the oligomerization status of overexpressed wild-type Cav1 and P132L with GFP, mCherry or myc tags by using this approach. In untransfected cells (Figure 3A–C, lane 1) or cells expressing either EGFP or mCherry alone (Figure 3A,B, lane 2) as a negative control, endogenous Cav1 ran as a high molecular weight band of ∼600 kDa. In contrast, cells expressing Cav1-GFP and Cav1-mCherry, two discrete high molecular weight bands were observed. The first had a similar mobility to that of endogenous Cav1 and was not recognized by an anti-GFP antibody (Figure 3A,B, lane 3, black arrow). Thus, it likely represents the endogenous Cav1 complex. The second band (Figure 3A,B, lane 3, red arrow) corresponded to a higher molecular weight and was positive for GFP or mCherry, suggesting that it consists of oligomers of Cav1-GFP or Cav1-mCherry. A smear of Cav1- and FPs-positive staining was also seen at lower and higher molecular weights than the band (Figure 3A,B, lane 3, brackets). This suggests that Cav1-GFP and Cav1-mCherry can form discrete high molecular weight oligomers distinct from those containing only endogenous Cav1, as well as oligomers of irregular size. Some FP-positive bands were seen at lower molecular weights that appear to consist of partially degraded forms of Cav1-GFP and Cav1-mCherry (Figure 3A,B, lane 3, green arrows). In contrast, overexpressed Cav1-myc formed a very wide band that overlapped with the position of endogenous Cav1 (Figure 3C, lane 2).


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

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

The oligomerization state of overexpressed Cav1 varies as a function of its tag. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for Cav1 (red) and either GFP, mCherry or myc (green). A) Cells were either left untransfected (‘control’) or transfected with EGFP, Cav1-GFP or P132L-GFP. B) As in (A) except cells were transfected with the indicated mCherry constructs. C) As in (A) except cells were transfected with Cav1-myc or P132L-myc. Figures are representative of two independent experiments. Red arrows indicate the high molecular weight band positive for both tag antibodies and Cav1 antibodies (h1-97 or 2297). Black arrows indicate the high molecular weight band only positive for Cav1 antibodies (h1-97 or 2297). Green arrows indicate the low molecular weight bands only positive for FP tag antibodies.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig03: The oligomerization state of overexpressed Cav1 varies as a function of its tag. COS-7 cells expressing the indicated constructs were lysed in digitonin and subjected to BN-PAGE followed by western blotting for Cav1 (red) and either GFP, mCherry or myc (green). A) Cells were either left untransfected (‘control’) or transfected with EGFP, Cav1-GFP or P132L-GFP. B) As in (A) except cells were transfected with the indicated mCherry constructs. C) As in (A) except cells were transfected with Cav1-myc or P132L-myc. Figures are representative of two independent experiments. Red arrows indicate the high molecular weight band positive for both tag antibodies and Cav1 antibodies (h1-97 or 2297). Black arrows indicate the high molecular weight band only positive for Cav1 antibodies (h1-97 or 2297). Green arrows indicate the low molecular weight bands only positive for FP tag antibodies.
Mentions: We next tested for possible defects in the oligomerization status of overexpressed wild-type Cav1 and P132L with GFP, mCherry or myc tags by using this approach. In untransfected cells (Figure 3A–C, lane 1) or cells expressing either EGFP or mCherry alone (Figure 3A,B, lane 2) as a negative control, endogenous Cav1 ran as a high molecular weight band of ∼600 kDa. In contrast, cells expressing Cav1-GFP and Cav1-mCherry, two discrete high molecular weight bands were observed. The first had a similar mobility to that of endogenous Cav1 and was not recognized by an anti-GFP antibody (Figure 3A,B, lane 3, black arrow). Thus, it likely represents the endogenous Cav1 complex. The second band (Figure 3A,B, lane 3, red arrow) corresponded to a higher molecular weight and was positive for GFP or mCherry, suggesting that it consists of oligomers of Cav1-GFP or Cav1-mCherry. A smear of Cav1- and FPs-positive staining was also seen at lower and higher molecular weights than the band (Figure 3A,B, lane 3, brackets). This suggests that Cav1-GFP and Cav1-mCherry can form discrete high molecular weight oligomers distinct from those containing only endogenous Cav1, as well as oligomers of irregular size. Some FP-positive bands were seen at lower molecular weights that appear to consist of partially degraded forms of Cav1-GFP and Cav1-mCherry (Figure 3A,B, lane 3, green arrows). In contrast, overexpressed Cav1-myc formed a very wide band that overlapped with the position of endogenous Cav1 (Figure 3C, lane 2).

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