FIGURE 3.

CDDO-Im delays TGFβ receptor degradation and trafficking. A, C2C12, COS-7, or Mv1Lu cells were affinity-labeled with ¹²⁵I-TGFβ at 4 °C, cross-linked, and lysed (zero time control) or incubated at 37 °C for 2, 4, or 8 h prior to lysis. One hundred micrograms of cell lysates were then subjected to SDS-PAGE followed by autoradiography and PhosphorImager analysis. The bands representing TβRI or TβRII are indicated on the left side of each panel. Total receptor levels were quantified and graphed (right panel) as receptor levels (percentage of control) versus time (n = 3 ± S.D.). B, Mv1Lu cells stably expressing TβRII were incubated at 4 °C with biotinylated TGFβ (bTGFβ), followed by streptavidin Cy3 (red). The cells were then incubated at 37 °C in the absence (left; Control) or presence (right) of 1 μm CDDO-Im for 1 h. Cells were then fixed, permeabilized, and immunostained with anti-caveolin-1 (Cav-1; green) and anti-EEA1 (EEA1; blue) antibodies. Areas of interest (white boxes) are magnified for both the control and CDDO-Im-treated cells and are shown below each panel (insets). Representative cells shown indicate the co-localization of biotinylated TGFβ with either caveolin-1 or EEA1 indicated by yellow or magenta arrowheads, respectively. The contour (dotted line) of each cell was established using bright field images and overlaid on the DAPI nuclear stain. Representative micrographs from three experiments are shown. Bar, 10 μm.