FIG. 1.

IRS-1 and p110α, but not p110β, concentrate in insulin-induced actin structures. Serum-deprived (4 h) L6 myotubes were stimulated with or without 100 nM insulin for 10 min at 37°C, followed by fixation and permeabilization. (A) Images were acquired from three focal planes that are depicted in a schematic diagram (a). The ventral (V) focal plane is defined as 1 to 3 μm above the coverslip; the dorsal (D) focal plane is 4 to 6 μm above the coverslip. In response to insulin, the surface of the cell remodels into structures that protrude 2 to 3 μm above the dorsal focal plane of the cell. This is defined as the dorsal extension (DE) focal plane that is absent in basal cells. Gray scale images of the ventral surface to the dorsal surface of basal (b and c) L6 myotubes and the ventral surface to the dorsal extensions of insulin-treated (d to f) L6 myotubes stained with Oregon Green-conjugated phalloidin are shown. (B) IRS-1 (a and d) or p110 (b, c, e, and f) proteins were stained with specific polyclonal antibodies, followed by Alexa 488-conjugated secondary antibody, as described in Materials and Methods. Composite images of F-actin labeled with Alexa fluor 633-conjugated phalloidin and IRS-1 or p110 protein staining are presented (yellow indicates regions of colocalization). Immunofluorescence images for p110α (g) and p110β (h) were also acquired from cells that were stained with antibodies preincubated with a 10-fold molar excess of competitive blocking peptides. The images are representative of three experiments. Bar, 10 μm.