Genetic and chemical manipulation of caveolae.

<p>Immunofluorescence labeling of endogenous Cav3 expression (Alexa 660) was compared to the distribution of exogenous expression of Cav3-GFP in myotubes. (A) Images of Alexa 660 immunolabeled Cav3 shows focal planes at the bottom (near the coverslip), the middle and the top of the cell. Cav3 has a punctate distribution at the cell surface (indicated by –-) and a reticulate internal structure. (B) Exogenously expressed Cav3-GFP shows the same patterns of distribution. (C) Cav3-GFP expressing cells treated for 1 hr with 5 mM MβCD at 37°C makes the surface distribution homogeneous (indicated by –––) and the internal proteins become associated with large vesicular structures (indicated by O). The image stacks showing all z-sections are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072894#pone.0072894.s009" target="_blank">Movies S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072894#pone.0072894.s010" target="_blank">S2</a>, and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072894#pone.0072894.s011" target="_blank">S3</a>. (D) Cav3-miRNA inhibits endogenous expression of Cav3 protein as shown by Alexa 660 immunofluorescence. DIC images show several myotubes. The myotube indicated with * has high GFP expression and low endogenous Cav3 expression compared to untransfected myotubes in the image. (E) Cells transfected with Cav3-miRNA showed significantly lower endogenous Cav3 expression than untransfected cells. The red dashed line shows the average Alexa 660 intensity of untransfected cells labeled without the primary antibody.